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1.  Taxonomic Status, Phylogenetic Affinities and Genetic Diversity of a Presumed Extinct Genus, Paraisometrum W.T. Wang (Gesneriaceae) from the Karst Regions of Southwest China 
PLoS ONE  2014;9(9):e107967.
The karst regions in South China have an abundance of endemic plants that face high extinction risks. The Chinese Gesneriaceae endemic Paraisometrum mileense ( = Oreocharis mileensis), was presumed extinct for 100 years. After its re-discovery, the species has become one of five key plants selected by the Chinese forestry government to establish a new conservation category for plants with extremely small populations. For conservation purposes, we studied the phylogenetic and population genetic status of P. mileense at the three only known localities in Guangxi, Guizhou and Yunnan.
Methodology/Principal Findings
We collected 64 samples (52 species) of Oreocharis and 8 samples from three provinces of P. mileense and generated molecular phylogenies, and inferred that P. mileense represents a relatively isolated and derived taxonomic unit within Oreocharis. Phylogeographic results of 104 samples of 12 populations of P. mileense indicated that the populations in Yunnan have derived from those in Guangxi and Guizhou. Based on AFLP data, the populations were found to harbor low levels of genetic diversity (He = 0.118), with no apparent gradient across the species’ range, a restricted gene flow and significant isolation-by-distance with limited genetic differentiation among the populations across the three provinces (FST = 0.207, P<0.001). The 10 populations in Yunnan were found to represent two distinct lineages residing at different altitudes and distances from villages.
The low levels of genetic diversity found in P. mileense are perhaps a consequence of severe bottlenecks in the recent past. The distribution of the genetic diversity suggests that all populations are significant for conservation. Current in situ and ex situ measures are discussed. Further conservation actions are apparently needed to fully safeguard this conservation flagship species. Our work provides a model of an integrated study for the numerous endemic species in the karst regions with extremely small populations.
PMCID: PMC4176718  PMID: 25250568
2.  Genetic diversity and domestication origin of tea plant Camellia taliensis (Theaceae) as revealed by microsatellite markers 
BMC Plant Biology  2014;14:14.
Tea is one of the most popular beverages in the world. Many species in the Thea section of the Camellia genus can be processed for drinking and have been domesticated. However, few investigations have focused on the genetic consequence of domestication and geographic origin of landraces on tea plants using credible wild and planted populations of a single species. Here, C. taliensis provides us with a unique opportunity to explore these issues.
Fourteen nuclear microsatellite loci were employed to determine the genetic diversity and domestication origin of C. taliensis, which were represented by 587 individuals from 25 wild, planted and recently domesticated populations. C. taliensis showed a moderate high level of overall genetic diversity. The greater reduction of genetic diversity and stronger genetic drift were detected in the wild group than in the recently domesticated group, indicating the loss of genetic diversity of wild populations due to overexploitation and habitat fragmentation. Instead of the endangered wild trees, recently domesticated individuals were used to compare with the planted trees for detecting the genetic consequence of domestication. A little and non-significant reduction in genetic diversity was found during domestication. The long life cycle, selection for leaf traits and gene flow between populations will delay the emergence of bottleneck in planted trees. Both phylogenetic and assignment analyses suggested that planted trees may have been domesticated from the adjacent central forest of western Yunnan and dispersed artificially to distant places.
This study contributes to the knowledge about levels and distribution of genetic diversity of C. taliensis and provides new insights into genetic consequence of domestication and geographic origin of planted trees of this species. As an endemic tea source plant, wild, planted and recently domesticated C. taliensis trees should all be protected for their unique genetic characteristics, which are valuable for tea breeding.
PMCID: PMC3890520  PMID: 24405939
Camellia taliensis; Domestication; Genetic diversity; Microsatellite; Tea plant
3.  Comparative Chloroplast Genomes of Camellia Species 
PLoS ONE  2013;8(8):e73053.
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.
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.
PMCID: PMC3751842  PMID: 24009730
4.  Complete chloroplast genome of the genus Cymbidium: lights into the species identification, phylogenetic implications and population genetic analyses 
Cymbidium orchids, including some 50 species, are the famous flowers, and they possess high commercial value in the floricultural industry. Furthermore, the values of different orchids are great differences. However, species identification is very difficult. To a certain degree, chloroplast DNA sequence data are a versatile tool for species identification and phylogenetic implications in plants. Different chloroplast loci have been utilized for evaluating phylogenetic relationships at each classification level among plant species, including at the interspecies and intraspecies levels. However, there is no evidence that a short sequence can distinguish all plant species from each other in order to infer phylogenetic relationships. Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution.
The complete nucleotide sequences of eight individuals from a total of five Cymbidium species’ chloroplast (cp) genomes were determined using Illumina sequencing technology of the total DNA via a combination of de novo and reference-guided assembly. The length of the Cymbidium cp genome is about 155 kb. The cp genomes contain 123 unique genes, and the IR regions contain 24 duplicates. Although the genomes, including genome structure, gene order and orientation, are similar to those of other orchids, they are not evolutionarily conservative. The cp genome of Cymbidium evolved moderately with more than 3% sequence divergence, which could provide enough information for phylogeny. Rapidly evolving chloroplast genome regions were identified and 11 new divergence hotspot regions were disclosed for further phylogenetic study and species identification in Orchidaceae.
Phylogenomic analyses were conducted using 10 complete chloroplast genomes from seven orchid species. These data accurately identified the individuals and established the phylogenetic relationships between the species. The results reveal that phylogenomics based on organelle genome sequencing lights the species identification—organelle-scale “barcodes”, and is also an effective approach for studying whole populations and phylogenetic characteristics of Cymbidium.
PMCID: PMC3644226  PMID: 23597078
Chloroplast genome; Phylogenomics; Species identification; Organelle-scale barcodes; Phylogeny; Divergence hotspot
5.  Exploiting EST databases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L.) 
BMC Plant Biology  2010;10:278.
The castor bean (Ricinus communis L.), a monotypic species in the spurge family (Euphorbiaceae, 2n = 20), is an important non-edible oilseed crop widely cultivated in tropical, sub-tropical and temperate countries for its high economic value. Because of the high level of ricinoleic acid (over 85%) in its seed oil, the castor bean seed derivatives are often used in aviation oil, lubricants, nylon, dyes, inks, soaps, adhesive and biodiesel. Due to lack of efficient molecular markers, little is known about the population genetic diversity and the genetic relationships among castor bean germplasm. Efficient and robust molecular markers are increasingly needed for breeding and improving varieties in castor bean. The advent of modern genomics has produced large amounts of publicly available DNA sequence data. In particular, expressed sequence tags (ESTs) provide valuable resources to develop gene-associated SSR markers.
In total, 18,928 publicly available non-redundant castor bean EST sequences, representing approximately 17.03 Mb, were evaluated and 7732 SSR sites in 5,122 ESTs were identified by data mining. Castor bean exhibited considerably high frequency of EST-SSRs. We developed and characterized 118 polymorphic EST-SSR markers from 379 primer pairs flanking repeats by screening 24 castor bean samples collected from different countries. A total of 350 alleles were identified from 118 polymorphic SSR loci, ranging from 2-6 per locus (A) with an average of 2.97. The EST-SSR markers developed displayed moderate gene diversity (He) with an average of 0.41. Genetic relationships among 24 germplasms were investigated using the genotypes of 350 alleles, showing geographic pattern of genotypes across genetic diversity centers of castor bean.
Castor bean EST sequences exhibited considerably high frequency of SSR sites, and were rich resources for developing EST-SSR markers. These EST-SSR markers would be particularly useful for both genetic mapping and population structure analysis, facilitating breeding and crop improvement of castor bean.
PMCID: PMC3017068  PMID: 21162723

Results 1-5 (5)