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1.  The Complete Chloroplast Genome of Guadua angustifolia and Comparative Analyses of Neotropical-Paleotropical Bamboos 
PLoS ONE  2015;10(12):e0143792.
To elucidate chloroplast genome evolution within neotropical-paleotropical bamboos, we fully characterized the chloroplast genome of the woody bamboo Guadua angustifolia. This genome is 135,331 bp long and comprises of an 82,839-bp large single-copy (LSC) region, a 12,898-bp small single-copy (SSC) region, and a pair of 19,797-bp inverted repeats (IRs). Comparative analyses revealed marked conservation of gene content and sequence evolutionary rates between neotropical and paleotropical woody bamboos. The neotropical herbaceous bamboo Cryptochloa strictiflora differs from woody bamboos in IR/SSC boundaries in that it exhibits slightly contracted IRs and a faster substitution rate. The G. angustifolia chloroplast genome is similar in size to that of neotropical herbaceous bamboos but is ~3 kb smaller than that of paleotropical woody bamboos. Dissimilarities in genome size are correlated with differences in the lengths of intergenic spacers, which are caused by large-fragment insertion and deletion. Phylogenomic analyses of 62 taxa yielded a tree topology identical to that found in preceding studies. Divergence time estimation suggested that most bamboo genera diverged after the Miocene and that speciation events of extant species occurred during or after the Pliocene.
doi:10.1371/journal.pone.0143792
PMCID: PMC4668023  PMID: 26630488
2.  ycf1, the most promising plastid DNA barcode of land plants 
Scientific Reports  2015;5:8348.
A DNA barcode is a DNA fragment used to identify species. For land plants, DNA fragments of plastid genome could be the primary consideration. Unfortunately, most of the plastid candidate barcodes lack species-level resolution. The identification of DNA barcodes of high resolution at species level is critical to the success of DNA barcoding in plants. We searched the available plastid genomes for the most variable regions and tested the best candidates using both a large number of tree species and seven well-sampled plant groups. Two regions of the plastid gene ycf1, ycf1a and ycf1b, were the most variable loci that were better than existing plastid candidate barcodes and can serve as a barcode of land plants. Primers were designed for the amplification of these regions, and the PCR success of these primers ranged from 82.80% to 98.17%. Of 420 tree species, 357 species could be distinguished using ycf1b, which was slightly better than the combination of matK and rbcL. For the well-sampled representative plant groups, ycf1b generally performed better than any of the matK, rbcL and trnH-psbA. We concluded that ycf1a or ycf1b is the most variable plastid genome region and can serve as a core barcode of land plants.
doi:10.1038/srep08348
PMCID: PMC4325322  PMID: 25672218
3.  A chloroplast genomic strategy for designing taxon specific DNA mini-barcodes: a case study on ginsengs 
BMC Genetics  2014;15:138.
Background
Universal conventional DNA barcodes will become more and more popular in biological material identifications. However, in many cases such as processed medicines or canned food, the universal conventional barcodes are unnecessary and/or inapplicable due to DNA degradation. DNA mini-barcode is a solution for such specific purposes. Here we exemplify how to develop the best mini-barcodes for specific taxa using the ginseng genus (Panax) as an example.
Results
The chloroplast genome of P. notoginseng was sequenced. The genome was compared with that of P. ginseng. Regions of the highest variability were sought out. The shortest lengths which had the same discrimination powers of conventional lengths were considered the best mini-barcodes. The results showed that the chloroplast genome of P. notoginseng is 156,387 bp. There are only 464 (0.30%) substitutions between the two genomes. The intron of rps16 and two regions of the coding gene ycf1, ycf1a and ycf1b, evolved the quickest and served as candidate regions. The mini-barcodes of Panax turned out to be 60 bp for ycf1a at a discrimination power of 91.67%, 100 bp for ycf1b at 100%, and 280 bp for rps16 at 83.33%.
Conclusions
The strategy by searching the whole chloroplast genomes, identifying the most variable regions, shortening the focal regions for mini-barcodes are believed to be efficient in developing taxon-specific DNA mini-barcodes. The best DNA mini-barcodes are guaranteed to be found following this strategy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-014-0138-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12863-014-0138-z
PMCID: PMC4293818  PMID: 25526752
Panax; Chloroplast genome; DNA mini-barcode; ycf1
4.  Complete Chloroplast Genome of Sedum sarmentosum and Chloroplast Genome Evolution in Saxifragales 
PLoS ONE  2013;8(10):e77965.
Comparative chloroplast genome analyses are mostly carried out at lower taxonomic levels, such as the family and genus levels. At higher taxonomic levels, chloroplast genomes are generally used to reconstruct phylogenies. However, little attention has been paid to chloroplast genome evolution within orders. Here, we present the chloroplast genome of Sedum sarmentosum and take advantage of several available (or elucidated) chloroplast genomes to examine the evolution of chloroplast genomes in Saxifragales. The chloroplast genome of S. sarmentosum is 150,448 bp long and includes 82,212 bp of a large single-copy (LSC) region, 16.670 bp of a small single-copy (SSC) region, and a pair of 25,783 bp sequences of inverted repeats (IRs).The genome contains 131 unique genes, 18 of which are duplicated within the IRs. Based on a comparative analysis of chloroplast genomes from four representative Saxifragales families, we observed two gene losses and two pseudogenes in Paeonia obovata, and the loss of an intron was detected in the rps16 gene of Penthorum chinense. Comparisons among the 72 common protein-coding genes confirmed that the chloroplast genomes of S. sarmentosum and Paeonia obovata exhibit accelerated sequence evolution. Furthermore, a strong correlation was observed between the rates of genome evolution and genome size. The detected genome size variations are predominantly caused by the length of intergenic spacers, rather than losses of genes and introns, gene pseudogenization or IR expansion or contraction. The genome sizes of these species are negatively correlated with nucleotide substitution rates. Species with shorter duration of the life cycle tend to exhibit shorter chloroplast genomes than those with longer life cycles.
doi:10.1371/journal.pone.0077965
PMCID: PMC3799696  PMID: 24205047
5.  Sequencing Angiosperm Plastid Genomes Made Easy: A Complete Set of Universal Primers and a Case Study on the Phylogeny of Saxifragales 
Genome Biology and Evolution  2013;5(5):989-997.
Plastid genomes are an invaluable resource for plant biological studies. However, the number of completely sequenced plant plastid genomes is still small compared with the vast number of species. To provide an alternative generalized approach, we designed a set of 138 pairs of universal primers for amplifying (termed “short-range PCR”) and sequencing the entire genomes of the angiosperm plastid genomes. The universality of the primers was tested by using species from the basal to asterid angiosperms. The polymerase chain reaction (PCR) success rate was higher than 96%. We sequenced the complete chloroplast genome of Liquidambar formosana as an example using this method and compared it to the genomes independently determined by long-range PCR (from 6.3 kb to 13.3 kb) and next-generation sequencing methods. The three genomes showed that they were completely identical. To test the phylogenetic efficiency of this method, we amplified and sequenced 18 chloroplast regions of 19 Saxifragales and Saxifragales-related taxa, as a case study, to reconstruct the phylogeny of all families of the order. Phylograms based on a combination of our data, together with those from GenBank, clearly indicate three family groups and three single families within the order. This set of universal primers is expected to accelerate the accumulation of angiosperm plastid genomes and to make faster mass data collection of plastid genomes for molecular systematics.
doi:10.1093/gbe/evt063
PMCID: PMC3673619  PMID: 23595020
plastid genome; universal primers; Saxifragales
6.  Highly Variable Chloroplast Markers for Evaluating Plant Phylogeny at Low Taxonomic Levels and for DNA Barcoding 
PLoS ONE  2012;7(4):e35071.
Background
At present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species.
Methodology/Principal Findings
We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest) were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnSUGA-trnGUCC, petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnSUGA-trnGUCC, trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (π values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species.
Significance/Conclusions
Chloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve closely related species or genera in phylogenetic analyses, and for DNA barcoding.
doi:10.1371/journal.pone.0035071
PMCID: PMC3325284  PMID: 22511980

Results 1-6 (6)