Hong-Tao Li

Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH–psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1–92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9–79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle‐scale barcodes. Next‐generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high‐quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long‐range PCR and sequenced using next‐generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early‐diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome‐scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.

Comparative Chloroplast Genomes of Camellia Species

Background Camellia , comprising more than 200 species, is a valuable economic commodity due to its enormously popular commercial products: tea leaves, flowers, and high-quality edible oils. It is the largest and most important genus in the family Theaceae. However, phylogenetic resolution of the species has proven to be difficult. Consequently, the interspecies relationships of the genus Camellia are still hotly debated. Phylogenomics is an attractive avenue that can be used to reconstruct the tree of life, especially at low taxonomic levels. Methodology/Principal Findings Seven complete chloroplast (cp) genomes were sequenced from six species representing different subdivisions of the genus Camellia using Illumina sequencing technology. Four junctions between the single-copy segments and the inverted repeats were confirmed and genome assemblies were validated by PCR-based product sequencing using 123 pairs of primers covering preliminary cp genome assemblies. The length of the Camellia cp genome was found to be about 157kb, which contained 123 unique genes and 23 were duplicated in the IR regions. We determined that the complete Camellia cp genome was relatively well conserved, but contained enough genetic differences to provide useful phylogenetic information. Phylogenetic relationships were analyzed using seven complete cp genomes of six Camellia species. We also identified rapidly evolving regions of the cp genome that have the potential to be used for further species identification and phylogenetic resolution. Conclusions/Significance In this study, we wanted to determine if analyzing completely sequenced cp genomes could help settle these controversies of interspecies relationships in Camellia . The results demonstrate that cp genome data are beneficial in resolving species definition because they indicate that organelle-based “barcodes”, can be established for a species and then used to unmask interspecies phylogenetic relationships. It reveals that phylogenomics based on cp genomes is an effective approach for achieving phylogenetic resolution between Camellia species.

Isolation and characterization of 15 microsatellite markers from wild tea plant (Camellia taliensis) using FIASCO method

Camellia taliensis is one of the most important wild tea plants in China, especially in Yunnan Province. In this study, we described the development of 15 microsatellite markers from the genome of C. taliensis using the protocol of fast isolation by AFLP of sequences containing repeats (FIASCO). Polymorphism of each locus was assessed in 24 samples collected from six wild populations of C. taliensis. The average allele number of the microsatellites was four per locus, ranging from 2 to 7. The observed and expected heterozygosities varied from 0.076 to 0.5833 and from 0.1560 to 0.6917, respectively. Cross-species amplification in other three tea plants showed eleven of them holding promise for sister species. These polymorphic SSR markers would be useful tools for population genetics studies and assessing genetic variations to establish conservation strategy, molecular identification and molecular breeding on this tea plant and its allied species and varieties in section Thea genus Camellia.

Complete Plastid Genome Sequencing of Four Tilia Species (Malvaceae): A Comparative Analysis and Phylogenetic Implications

Tilia is an ecologically and economically important genus in the family Malvaceae. However, there is no complete plastid genome of Tilia sequenced to date, and the taxonomy of Tilia is difficult owing to frequent hybridization and polyploidization. A well-supported interspecific relationships of this genus is not available due to limited informative sites from the commonly used molecular markers. We report here the complete plastid genome sequences of four Tilia species determined by the Illumina technology. The Tilia plastid genome is 162,653 bp to 162,796 bp in length, encoding 113 unique genes and a total number of 130 genes. The gene order and organization of the Tilia plastid genome exhibits the general structure of angiosperms and is very similar to other published plastid genomes of Malvaceae. As other long-lived tree genera, the sequence divergence among the four Tilia plastid genomes is very low. And we analyzed the nucleotide substitution patterns and the evolution of insertions and deletions in the Tilia plastid genomes. Finally, we build a phylogeny of the four sampled Tilia species with high supports using plastid phylogenomics, suggesting that it is an efficient way to resolve the phylogenetic relationships of this genus.

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle‐scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long‐range polymerase chain reaction and using next‐generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel‐dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel‐dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.

Plastid Phylogenomic Analyses Resolve Tofieldiaceae as the Root of the Early Diverging Monocot Order Alismatales.

The predominantly aquatic order Alismatales, which includes approximately 4,500 species within Araceae, Tofieldiaceae, and the core alismatid families, is a key group in investigating the origin and early diversification of monocots. Despite their importance, phylogenetic ambiguity regarding the root of the Alismatales tree precludes answering questions about the early evolution of the order. Here, we sequenced the first complete plastid genomes from three key families in this order: Potamogeton perfoliatus (Potamogetonaceae), Sagittaria lichuanensis (Alismataceae), and Tofieldia thibetica (Tofieldiaceae). Each family possesses the typical quadripartite structure, with plastid genome sizes of 156,226, 179,007, and 155,512 bp, respectively. Among them, the plastid genome of S. lichuanensis is the largest in monocots and the second largest in angiosperms. Like other sequenced Alismatales plastid genomes, all three families generally encode the same 113 genes with similar structure and arrangement. However, we detected 2.4 and 6 kb inversions in the plastid genomes of Sagittaria and Potamogeton, respectively. Further, we assembled a 79 plastid protein-coding gene sequence data matrix of 22 taxa that included the three newly generated plastid genomes plus 19 previously reported ones, which together represent all primary lineages of monocots and outgroups. In plastid phylogenomic analyses using maximum likelihood and Bayesian inference, we show both strong support for Acorales as sister to the remaining monocots and monophyly of Alismatales. More importantly, Tofieldiaceae was resolved as the most basal lineage within Alismatales. These results provide new insights into the evolution of Alismatales as well as the early-diverging monocots as a whole.

Isolation and characterization of 15 microsatellite markers from the spring orchid (Cymbidium goeringii) (Orchidaceae).

PREMISE OF THE STUDY Microsatellite primers were developed for the important and popular ornamental spring orchid, Cymbidium goeringii, to investigate its genetic diversity and population genetic structure. METHODS AND RESULTS Using the Fast Isolation by AFLP of Sequences Containing (FIASCO) repeats protocol, 15 primer sets were identified in two wild populations. The number of alleles per locus ranged from 2 to 5, with a mean of 3.3. The observed and expected heterozygosities varied from 0.167 to 0.917 and from 0.159 to 0.822, respectively. All these primers successfully amplified in the congener C. tortisepalum, and 12 primers were found useful in C. faberi and C. sinense. CONCLUSIONS These markers will facilitate further studies on the population genetics of Cymbidium goeringii and other congeneric species.

A molecular phylogenetic study of Hemsleya (Cucurbitaceae) based on ITS, rpl16, trnH-psbA, and trnL DNA sequences

This paper presents the first molecular phylogeny of the genus Hemsleya using nuclear ITS and plastid trnH-psbA, rpl16, and trnL DNA sequences to examine the relationships among Hemsleya species. Phylogenetic relationships were elucidated using a combined analysis of all four datasets, however, the number of parsimony-informative characters was still insufficient to resolve all relationships. Parsimony and Bayesian trees were highly congruent. Twenty-three species of Hemsleya split into two major clades corresponding to two subgenera, i.e., subg. Graciliflorae and subg. Hemsleya. These results are partly in agreement with Li’s sectional classification. However, the molecular data are inconsistent with Li’s classification at the subsectional level. The molecular phylogeny revealed a striking overall correlation between the phylogenetic relationships of the species and their geographical distribution. The Kangdian ancient landmass could be the center of origin of the genus.

Phylogeny and Evolution of Bracts and Bracteoles in Tacca (Dioscoreaceae)

Most species in the genus Tacca (Dioscoreaceae) feature green to black purple, conspicuous inflorescence involucral bracts with variable shapes, motile filiform appendages (bracteoles), and diverse types of inflorescence morphology. To infer the evolution of these inflorescence traits, we reconstructed the molecular phylogeny of the genus, using DNA sequences from one nuclear, one mitochondrial, and three plastid loci (Internal Transcribed Spacer (ITS), atpA, rbcL, trnL-F, and trnH-psbA). Involucres and bracteoles characters were mapped onto the phylogeny to analyze the sequence of inflorescence trait evolution. In all analyses, species with showy involucres and bracteoles formed the most derived clade, while ancestral Tacca had small and plain involucres and short bracteoles, namely less conspicuous inflorescence structures. Two of the species with the most elaborate inflorescence morphologies (T. chantrieri in southeast China and T. integrifolia in Tibet), are predominantly self-pollinated, indicating that these conspicuous floral displays have other functions rather than pollinator attraction. We hypothesize that the motile bracteoles and involucres may facilitate selfing; display photosynthesis in the dim understory, and protect flowers from herbivory.