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. 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 trnS^UGA-trnG^UCC, petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnS^UGA-trnG^UCC, 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. 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.

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.

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.

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. 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%. 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.

Walnuts (Juglans of the Juglandaceae) are well-known economically important resource plants for the edible nuts, high-quality wood, and medicinal use, with a distribution from tropical to temperate zones and from Asia to Europe and Americas. There are about 21 species in Juglans. Classification of Juglans at section level is problematic, because the phylogenetic position of J. cinerea is disputable. Lacking morphological and DNA markers severely inhibited the development of related researches. In this study, the complete chloroplast genomes and two nuclear DNA regions (the internal transcribed spacer and ubiquitin ligase gene) of ten representative taxa of Juglans were used for comparative genomic analyses in order to deepen the understanding on the application value of genetic information for inferring the phylogenetic relationship of the genus. The Juglans chloroplast genomes possessed the typical quadripartite structure of angiosperms, consisting of a pair of inverted repeat regions separated by a large single-copy region and a small single-copy region. All the ten chloroplast genomes possessed 112 unique genes arranged in the same order, including 78 protein-coding, 30 tRNA, and 4 rRNA genes. A combined sequence data set from two nuclear DNA regions revealed that Juglans plants could be classified into three branches: (1) section Juglans, (2) section Cardiocaryon including J. cinerea which is closer to J. mandshurica, and (3) section Rhysocaryon. However, three branches with a different phylogenetic topology were recognized in Juglans using the complete chloroplast genome sequences: (1) section Juglans, (2) section Cardiocaryon, and (3) section Rhysocaryon plus J. cinerea. The molecular taxonomy of Juglans is almost compatible to the morphological taxonomy except J. cinerea (section Trachycaryon). Based on the complete chloroplast genome sequence data, the divergence time between section Juglans and section Cardiocaryon was 44.77 Mya ago, while section Rhysocaryon diverged from other sections in the genus Juglans was 47.61 Mya ago. Eleven of the twelve small inversions in the chloroplast genomes provided valuable phylogenetic information for classification of walnut plants at section and species levels. Our results are valuable for future studies on Juglans genetic diversity and will enhance the understanding on the phylogenetic evolution of Juglandaceae.

Machilus is a large (c. 100 spp.) genus of trees in the family Lauraceae, distributed in tropical and subtropical East Asia. Both molecular species identification and phylogenetic studies of this morphologically uniform genus have been constrained by insufficient variable sites among frequently-used biomarkers. To better understand the mutation patterns in the chloroplast genome of Machilus, the complete plastomes of two species were sequenced. The plastomes of M. yunnanensis and M. balansae were 152, 622 bp and 152, 721 bp respectively. Seven highly variable regions between the two Machilus species and three of them between Machilus and Cinnamomum were identified and 297 mutation events, including one micro-inversion in the ccsA-ndhD region, 65 indels, and 231 substitutions, were accurately located. Thirty-six microsatellite sites were found for use in species identification and 95 single-nucleotide changes were identified in gene coding regions.