Testing four candidate barcoding markers in temperate woody bamboos (Poaceae: Bambusoideae)

Abstract Bambusoideae is an important subfamily of the grass family Poaceae that has considerable economic, ecologic and cultural value. In addition, Bambusoideae species are important constituents of the forest vegetation in China. Because of the paucity of flower‐bearing specimens and homoplasies of morphological characters, it is difficult to identify species of Bambusoideae using morphology alone, especially in the case of temperate woody bamboos (i.e. Arundinarieae). To this end, DNA barcoding has shown great potential in identifying species. The present study is the first attempt to test the feasibility of four proposed DNA barcoding markers (matK, rbcL, trnH–psbA, and internal transcribed spacer [ITS]) in identifying 27 species of the temperate woody bamboos. Three plastid markers showed high levels of universality, whereas the universality of ITS was comparatively low. A single plastid marker provided low levels of discrimination success at both the genus and species levels (<12%). Among the combinations of plastid markers, the highest discriminatory power was obtained using the combination of rbcL+matK (14.8%). Using a combination of three markers did not increase species discrimination. The nuclear region ITS alone could identify 66.7% of species, although fewer taxa were included in the ITS analyses than in the plastid analyses. When ITS was integrated with a single or combination of plastid markers, the species discriminatory power was significantly improved. We suggest that a combination of rbcL+ ITS, which exhibited the highest species identification power of all combinations in the present study, could be used as a potential DNA barcode for temperate woody bamboos.     

Molecular identification of species in Juglandaceae:A tiered method

DNA barcoding is a method of species identification and recognition using DNA sequence data. A tiered or multilocus method has been recommended for barcoding plant species. In this study, we sampled 196 individuals representing 9 genera and 54 species of Juglandaceae to investigate the utility of the four potential barcoding loci (rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS)). Our results show that all four DNA regions are easy to amplify and sequence. In the four tested DNA regions, ITS has the most variable information, and rbcL has the least. At generic level, seven of nine genera can be efficiently identified by matK. At species level, ITS has higher interspecific p-distance than the trnH-psbA region. Difficult to align in the whole family, ITS showed heterogeneous variability among different genera. Except for the monotypic genera (Cyclocarya, Annamocarya, Platycarya), ITS appeared to have limited power for species identification within the Carya and Engelhardia complex, and have no power for Juglans or Pterocarya. Overall, our results confirmed that a multilocus tiered method for plant barcoding was applicable and practicable. With higher priority, matK is proposed as the first-tier DNA region for genus discrimination, and the second locus at species level should have enough stable variable characters.     

New universal matK primers for DNA barcoding angiosperms

The chloroplast maturase K gene (matK) is one of the most variable coding genes of angiosperms and has been suggested to be a "barcode" for land plants. However, matK exhibits low amplification and sequencing rates due to low universality of currently available primers and mononucleotide repeats. To resolve these technical problems, we evaluated the entire matK region to find a region of 600-800 bp that is highly variable, represents the best of all matK regions with priming sites conservative enough to design universal primers, and avoids the mononucleotide repeats. After careful evaluation, a region in the middle was chosen and a pair of primers named natK472F and matK1248R was designed to amplify and sequence the matK fragment of approximately 776 bp. This region encompasses the most variable sites, represents the entire matK region best, and also exhibits high amplification rates and quality of sequences. The universality of this primer pair was tested using 58 species from 47 families of angiosperm plants. The primers showed a strong amplification (93.1%) and sequencing (92.6%)successes in the species tested. We propose that the new primers will solve, in part, the problems encountered when using matK and promote the adoption of matK as a DNA barcode for angiosperms.     

基于matK基因的松属白皮松组分子系统发育分析

基于matK基因对松属(Pinus L.)白皮松组(sect.Parrya Myre)进行了分子系统发育分析.白皮松组为一个并系类群,因为白松组的成员与该组(包括越南的扁叶松(P.krempfii Lecomte))的亚洲成员形成一个强烈支持的分支(靴带值92%).在这个分支中,白松组的3个代表种形成一个稳定的单系,而白皮松组的亚洲成员之间系统发育关系不明确.扁叶松和西藏白皮松(P. gerardiana Wall.ex D.Don)聚在一起,但只有61%的支持率.虽然在以前4个cpDNA基因序列分析时五针白皮松(P.squamata X.W.Li)与白皮松(P.bungeana Zucc.ex Loud.)和西藏白皮松形成一个单系,但在本文的分析中三者的关系不明确.在邻接树和多数一致简约树上,北美的白皮松组成员形成一个支持率低的分支.北美的subsect.Balfourianae Engelm.亚组(包括P.aristata Engelm.)是一个单系,但支持率较低.美洲另外两个亚组subsect.Cembroides Englem.和subsect.Rzedowskianae Carv.的组间和组内关系不确定,它们在严格一致简约树上形成一个多歧分支.     

DNA barcoding,microarrays and next generation sequencing: recent tools for genetic diversity estimation and authentication of medicinal plants

DNA barcoding, microarray technology and next generation sequencing have emerged as promising tools for the elucidation of plant genetic diversity and its conservation. They are proving to be immensely helpful in authenticating the useful medicinal plants for herbal drug preparations. These newer versions of molecular markers utilize short genetic markers in the genome to characterize the organism to a particular species. This has the potential not only to classify the known and yet unknown species but also has a promising future to link the medicinally important plants according to their properties. The newer trends being followed in DNA chips and barcoding pave the way for a future with many different possibilities. Several of these possibilities might be: characterization of unknown species in a considerably less time than usual, identification of newer medicinal properties possessed by the species and also updating the data of the already existing but unnoticed properties. This can assist us to cure many different diseases and will also generate novel opportunities in medicinal drug delivery and targeting.     

Molecular evidence for polyploid origins in Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies

The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis: (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua. Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis. The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin.     

Phylogenetic relationships of Cornaceae and close relatives inferred from matK and rbcL sequences

Phylogenetic relationships were inferred using nucleotide sequences of the chloroplast gene matK for members of Cornales, a well-supported monophyletic group comprising Cornaceae and close relatives. The shortest trees resulting from this analysis were highly concordant with those based on previous phylogenetic analysis of rbcL sequences. Analysis of a combined matK and rbcL sequence data set (a total of 2652 bp [base pairs]) provided greater resolution of relationships and higher internal support for clades compared to the individual data sets. Four major clades (most inclusive monophyletic groups) of Cornales are indicated by both sets of genes: (1) Cornus-Alangium, (2) nyssoids (Nyssa-Davidia-Camptotheca)- mastixioids (Mastixia, Diplopanax), (3) Curtisia, and (4) Hydrangeaceae-Loasaceae. The combined evidence indicates that clades 2 and 3 are sisters, with clade 4 sister to the remainder of Cornales. These relationships are also supported by other lines of evidence, including synapomorphies in fruit and pollen morphology and gynoecial vasculature. Comparisons of matK and rbcL sequences based on one of the most parsimonious rbcL-matK trees indicate that matK has a much higher A-T content (66.9% in matK vs. 55.8% in rbcL) and a lower transition:transversion ratio (1.23 in matK vs. 2.21 in rbcL). The total number of nucleotide substitutions per site for matK is 2.1 times that of rbcL in Cornales. These findings are similar to recent comparisons of matK and rbcL in other dicots. Variable sites of matK are almost evenly distributed among the three codon positions (1.0:1.0:1.3), whereas variable sites of rbcL are mostly at the third position (1.8:1.0 :7.5). Among- lineages rates of nucleotide substitutions in rbcL are basically homogeneous throughout Cornales, but are more heterogeneous in matK.     

Relationships within Cupressaceae sensu lato: a combined morphological and molecular approach

Parsimony analysis of matK and rbcL sequence data, together with a nonmolecular database, yielded a well-resolved phylogeny of Cupressaceae sensu lato. Monophyly of Cupressaceae sensu stricto is well supported, and separate northern and southern hemisphere subclades are resolved, with Tetraclinis within the northern subclade; there is no support for any of the tribes sensu Li. Taxodiaceae comprise five separate lineages. Chamaecyparis nootkatensis falls within Cupressus, clustering with a robust clade of New World species. Libocedrus Florin is paraphyletic and should incorporate Pilgerodendron. Evolution of several characters of wood and leaf anatomy and chemistry is discussed in light of this estimate of the phylogeny; numerous parallelisms are apparent. A new infrafamilial classification is proposed in which seven subfamilies are recognized: Callitroideae Saxton, Athrotaxidoideae Quinn, Cunninghamioideae (Sieb. & Zucc.) Quinn, Cupressoideae Rich. ex Sweet, Sequoioideae (Luerss.) Quinn, Taiwanioideae (Hayata) Quinn, Taxodioideae Endl. ex K. Koch. The rbcL sequence for Taxodium distichum is corrected, and the implications for a previously published estimate of the minimum rate of divergence of the gene since the Miocene are highlighted.