Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding

DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254–767 bp) and of a shorter fragment of this intron (the P6 loop, 10–143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.     

New insights on diet variability revealed by DNA barcoding and high-throughput pyrosequencing: chamois diet in autumn as a case study

Characterizing the diet of large herbivores and the determinants of its variation remains a difficult task in wild species. DNA-based techniques have the potential to complement traditional time-consuming methods based on the microhistology of plant cuticle fragments in fecal or rumen samples. Recently, it has been shown that a short chloroplast DNA fragment, the P6 loop of the trnL (UAA) intron, can act as a minimalist barcode. Here, we used the trnL approach with high-throughput pyrosequencing to study diet from feces in a wild herbivore, the alpine chamois (Rupicapra rupicapra) and showed that the fine resolution in plant determination obtained with this method allows exploring subtle temporal shifts and inter-individual variability in diet composition. First, we built a DNA barcoding database of 475 plants species. Seventy-two percent of plant species can be unambiguously identified to species level, 79% to genus level and 100% to family level using the P6 loop. Second, we analysed 74 feces collected from October to November. Based on 47,896 P6 loop sequences, we identified a total of 110 taxa, 96 in October and 76 in November, with a clear diet shift between October and November. We recognized four and two clusters of feces composition in October and November, respectively, revealing different diet categories among individuals within each month. DNA-based diet analysis is faster and more taxonomically precise than studies based on microhistology, and opens new possibilities for analysing plant-herbivore interactions in the wild.     

The determination of multiple microsatellite genotypes and DNA sequences from a single pollen grain

Pollen plays important roles in the reproduction and gene flow of flowering plants, and its haploid DNA sequence provides useful information for studies of plant evolution and genealogy. We describe a new method for multiple microsatellite genotyping and DNA sequencing from a single pollen grain. The haploid DNA was extracted from a single pollen grain by using a simple DNA extraction method, and multiple microsatellite genotypes and DNA sequences of multiple chloroplast loci were determined. Using nine pairs of microsatellite primers, more than 90% of genotypes were successfully determined, and 71% and 100% of DNA sequences were determined at two chloroplast DNA loci, the trnL intron region and the trnL/trnF intergenic spacer region, respectively. This simple method of genetic analysis for a single pollen grain will facilitate detailed study of pollination, evolution and genealogy.     

Amplicon pyrosequencing late Pleistocene permafrost: the removal of putative contaminant sequences and small‐scale reproducibility

DNA sequencing of ancient permafrost samples can be used to reconstruct past plant, animal and bacterial communities. In this study, we assess the small‐scale reproducibility of taxonomic composition obtained from sequencing four molecular markers (mitochondrial 12S ribosomal DNA (rDNA), prokaryote 16S rDNA, mitochondrial cox1 and chloroplast trnL intron) from two soil cores sampled 10 cm apart. In addition, sequenced control reactions were used to produce a contaminant library that was used to filter similar sequences from sample libraries. Contaminant filtering resulted in the removal of 1% of reads or 0.3% of operational taxonomic units. We found similar richness, overlap, abundance and taxonomic diversity from the 12S, 16S and trnL markers from each soil core. Jaccard dissimilarity across the two soil cores was highest for metazoan taxa detected by the 12S and cox1 markers. Taxonomic community distances were similar for each marker across the two soil cores when the chi‐squared metric was used; however, the 12S and cox1 markers did not cluster well when the Goodall similarity metric was used. A comparison of plant macrofossil vs. read abundance corroborates previous work that suggests eastern Beringia was dominated by grasses and forbs during cold stages of the Pleistocene, a habitat that is restricted to isolated sites in the present‐day Yukon.     

DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples

Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. Sample preparation and DNA extraction are of fundamental importance for obtaining an optimal DNA yield. Currently, there is no standard procedure for these steps, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols for processing environmental samples for pollen DNA extraction and for metabarcoding analysis and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen collected by gravimetric (Tauber trap) and volumetric (Hirst-type trap) samplers. Protocols were tested across an increasing complexity of samples, from pure single-species pollen to environmental multi-species samples. A short fragment (about 150 base pairs) of the chloroplast trnL gene was amplified using universal primers for plants. After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison with a custom-made reference database including chloroplast DNA sequences from most of the anemophilous taxa occurring in the study area (Trentino, northern Italy), representing 46 plant families. Using the classical morphological pollen analysis as a benchmark, we show that DNA metabarcoding is efficient and applicable even in complex samples, provided that protocols for sample preparation, DNA extraction, and metabarcoding analysis are carefully optimized.     

A deep dig––hindsight on Holocene vegetation composition from ancient environmental DNA

Want a glimpse at past vegetation? Studying pollen and other plant remains, which are preserved for example in lake sediments or mires for thousands of years, allows us to document regional occurrences of plant species over radiocarbon‐dated time series. Such vegetation reconstructions derived from optical analyses of fossil samples are inherently incomplete because they only comprise taxa that contribute sufficient amounts of pollen, spores, macrofossil or other evidences. To complement optical analyses for paleoecological inference, molecular markers applied to ancient DNA (aDNA) may help in disclosing information hitherto inaccessible to biologists. Parducci et al. (2013) targeted aDNA from sediment cores of two lakes in the Scandes Mountains with generic primers in a meta‐barcoding approach. When compared to palynological records from the same cores, respective taxon lists show remarkable differences in their compositions, but also in quantitative representation and in taxonomic resolution similar to a previous study (Jørgensen et al. 2012). While not free of assumptions that need critical and robust testing, notably the question of possible contamination, this study provides thrilling prospects to improve our knowledge about past vegetation composition, but also other organismic groups, stored as a biological treasure in the ground.     

Diet analysis by next‐generation sequencing indicates the frequent consumption of introduced plants by the critically endangered red‐headed wood pigeon (Columba janthina nitens) in oceanic island habitats

Oceanic island ecosystems are vulnerable to the introduction of alien species, and they provide a habitat for many endangered species. Knowing the diet of an endangered animal is important for appropriate nature restoration efforts on oceanic islands because introduced species may be a major component of the diets of some endangered species. DNA barcoding techniques together with next‐generation sequencing may provide more detailed information on animal diets than other traditional methods. We performed a diet analysis using 48 fecal samples from the critically endangered red‐headed wood pigeon that is endemic to the Ogasawara Islands based on chloroplast trnL P6 loop sequences. The frequency of each detected plant taxa was compared with a microhistological analysis of the same sample set. The DNA barcoding approach detected a much larger number of plants than the microhistological analysis. Plants that were difficult to identify by microhistological analysis after being digested in the pigeon stomachs were frequently identified only by DNA barcoding. The results of the barcoding analysis indicated the frequent consumption of introduced species, in addition to several native species, by the red‐headed wood pigeon. The rapid eradication of specific introduced species may reduce the food resources available to this endangered bird; thus, balancing eradication efforts with the restoration of native food plants should be considered. Although some technical problems still exist, the trnL approach to next‐generation sequencing may contribute to a better understanding of oceanic island ecosystems and their conservation.     

Phylogenetic Analysis of AA-genome Oryza Species (Poaceae) Based on Chloroplast, Mitochondrial, and Nuclear DNA Sequences

Species in the genus Oryza (Poaceae) contain 10 genomic types and are distributed in pan-tropics of the world. To explore phylogenetic relationships of Oryza species having the AA-genome, DNA sequences of the chloroplast trnL intron and trnL-trnF spacer, mitochondrial nad1 intron 2, and nuclear internal transcribed spacer were analyzed, based on materials from 6 cultivated (O. sativa and O. glaberrima) and 13 wild accessions, in addition to a CC-genome species (O. officinalis) that was used as an outgroup. Analyses of the combined sequence data set from different sources provide a much better resolution of the AA-genome species than the individual data set, indicating the limitation of a single gene in phylogenetic reconstruction. The phylogeny based on the combined data set demonstrated an apparent grouping of the AA-genome Oryza species that was well associated with their geographic origin, although the Australian O. meridionalis showed its affinity with the African species. The geographic pattern of the phylogenetic relationship was probably attributed to the frequent genetic exchange and introgression among the AA-genome species from the same continents. In addition, Asian cultivated rice O. sativa showed its close relation to O. rufipogon and O. nivara, whereas African cultivated rice O. glaberrima was closely linked to O. barthii and O. longistaminata, indicating the independent domestication of the two cultivated species in different geographic locations.     

Genetic diversity of dodder (<Emphasis Type="Italic">Cuscuta</Emphasis> spp.) collected from commercial cranberry production as revealed in the<Emphasis Type="Italic">trn</Emphasis>L (UAA) intron

Swamp dodder (Cuscuta gronovii) is a parasitic plant detrimental to cranberries. Observation of emergence of dodder seeds collected from a cultivated cranberry bog in Massachusetts revealed 2 or more peak emergence times during 4 consecutive growing seasons. Molecular methods were used to investigate genetic variation among the emerging dodder seedlings. On emergence, dodder seedlings were collected and analyzed for DNA sequence diversity in thetrnL (UAA) intron, a noncoding region of chloroplast DNA. DNA sequence analysis of 87 seedlings collected during the 1999 and 2000 growing seasons revealed the presence of 2 dodder ecotypes, designated A and B. Comparative DNA sequence analysis indicated that in thetrnL (UAA) intron, the sequence of ecotype A is identical to that ofCuscuta gronovii, whereas the sequence of ecotype B is closest to that ofCuscuta attenuata (99.3% sequence identity; 293 bases considered). ABg/II restriction enzyme cut site was identified that distinguished between thetrnL (UAA) introns of ecotypes A and B. Restriction fragment length polymorphism (RFLP) was used to analyze the sequences of 100 seedlings collected during the growing seasons of 2001 and 2002. Only 10 of the 187 samples were ecotype A, all of which emerged on or before May 7 in the growing seasons. Therefore, the predominant dodder haplotype found in this study may be a close relative ofC. attenuata and notC. gronovii, the common species found in cranberry bogs.     

TrnL–trnF Intergenic Spacer and trnL Intron Define Major Clades Within Luzula and Juncus (Juncaceae): Importance of Structural Mutations

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL–trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion o within a pseudogene) within the chloroplast trnL–trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The Southern Hemisphere clade was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5 acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL–trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.     

Phylogenetic Use of Noncoding Regions in the GenusGentianaL.: ChloroplasttrnL (UAA) Intron versus Nuclear Ribosomal Internal Transcribed Spacer Sequences

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA)trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genusGentianaL. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in thetrnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least inGentiana,ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNAtrnL intron seems to be preferable at the intergeneric level.     

Phylogenetic relationships between disjunctly occurring groups of Tristicha trifaria (Podostemaceae)

Aim To reveal the phylogeographic relationship of disjunct specimens of Tristicha trifaria (Bory ex Willd.) Spreng., a member of the Podostemaceae river‐weed family, which is distributed exceptionally widely, but disjunctly, in Africa and the Americas. Location Brazil, Mexico, Ghana, Tanzania and Madagascar. Methods The chloroplast matK and rbcL genes, a trnK intron, the trnS‐trnG intergenic spacer (IGS), the two IGSs of trnT‐trnL‐trnF, a trnL intron, and nuclear ribosomal ITS regions were sequenced and analysed. Phylogenetic analyses were conducted using maximum likelihood and maximum parsimony methods. Results The T. trifaria samples analysed were separated into two groups in a rooted tree based on a combined matK/rbcL/ITS dataset; one contained the West African and all of the American samples, and the other contained the East African and Madagascan samples. An unrooted tree obtained from a combined analysis of all the chloroplast DNA and nuclear ITS data showed that a sample from West Africa was sister to an American T. trifaria group. Main conclusions The American and West African T. trifaria are closely related, despite the great distance between their locations. This observation, along with a tree of the whole Tristichoideae subfamily and estimated divergence times, suggests that an ancestor of T. trifaria migrated from Asia to Africa during the early Tertiary, and that this was followed by further westward migration to the Americas at the end of the Miocene or in the early Pliocene.     

利用DNA片段测序方法探究枳属和富民枳的分类地位

富民枳(Poncirus polyandra)属于芸香科(Rutaceae)枳属(Poncirus Raf.).自发表以来,分类地位一直备受争议,其中在Flora of China中认为富民枳为柑橘杂交种(Poncirus polyandra),把枳属归并于柑橘属(Citrus).该研究选取枳属的富民枳、枳(Ponci...     

Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA

A protocol using insect specimens or parts thereof allows for sequencing of sections of nuclear 28S rDNA. In the present note it is demonstrated that this protocol can readily be applied to strongly degraded DNA (ancient, fixed or contaminated). Primers that are specifically designed to discriminate against human DNA but also other non‐arthropod species are tested on a range of species covering all insect groups (59 insect species from all 33 orders). Additionally, the samples represent a selection of various, mostly DNA‐degrading, preservation methods, including the most common fixatives used for morphological investigations and for long‐term storage in collections. Successful amplification was possible for all tested samples including ca. 200 year‐old dried museum specimens as well as for over 4000 year‐old fossil insects embedded in copal. When the NCBI database contained information on the tested species an unambiguous taxonomic discrimination was possible. This approach is based on a standardized protocol that guarantees easy application. This note presents primer pairs for 28S rDNA that can be a useful tool for ancient DNA (aDNA) research.     

DNA sequences of tobacco chloroplast genes for tRNASer (GGA), tRNAThr (UGU), tRNALeu (UAA), tRNAPhe (GAA): the tRNALeu gene contains a 503 bp intron

Summary The location and nucleotide sequence of tobacco chloroplast genes for tRNA^Ser (GGA), tRNA^Thr (UGU), tRNA^Leu (UAA) and tRNA^Phe (GAA) (trnS-GGA, trnT-UGU, trnL-UAA and trnF-GAA, respectively) have been determined. These genes are located in the 10 kbp BamHI fragment which lies in the middle of the large single-copy region of the chloroplast DNA. The gene order is trnS-trnT-trnL-trnF. The trnS, trnL and trnF are encoded on the same strand while the trnT on the opposite strand. The trnL contains a 503 bp intron like maize and broad bean trnL-UAAs.     

Lack of trnL intron in the thermoacidophilic red alga Galdieria sulphuraria (Galdieri) Merola

Abstract

The plastid trnC‐trnL(UAA)‐ilvH region from Galdieria sulphuraria was cloned and sequenced with the aim of verifying the absence of the trnL intron. The sequence alignment shows both the absence of a trnL intron and the colinearity of the whole region of the plastidial DNA of G. sulphuraria with that of the other thermoacidophilic red algae.     

Ancient DNA from pollen: a genetic record of population history in Scots pine

Assessments of plant population dynamics in space and time have depended on dated records of fossil pollen synthesized on a subcontinental scale. Genetic analyses of extant populations have revealed spatial relationships that are indicative of past spatial dynamics, but lack an explicit timescale. Synthesis of these data requires genetic analyses from abundant dated fossil material, and this has hitherto been lacking. Fossil pollen is the most abundant material with which to fill this data gap. Here we report genetic analyses of fossil pollen retrieved from Holtjärnen postglacial lake sediment in Sweden and show that plastid DNA is recoverable from Scots Pine and Norway spruce pollen grains that are 100 and 10 000 years old. By sequencing clones from two short plastid PCR products and by using multiple controls we show that the ancient sequences were endogenous to the fossil grains. Comparison of ancient sequences and those obtained from an extant population of Scots pine establishes the first genetic link between extant and fossil samples in this species, providing genetic continuity through time. The finding of one common haplotype present in modern, 100-year old and 10 000-year old samples suggests that it may have persisted near Holtjärnen throughout the postglacial period. This retrieval of ancient DNA from pollen has major implications for plant palaeoecology in conifer species by allowing direct estimates of population dynamics in space and time.     

Efficient and sensitive identification and quantification of airborne pollen using next‐generation DNA sequencing

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen‐allergic patients. Current pollen monitoring methods are microscope‐based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost‐effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next‐generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.     

Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants

Abstract Species of Prunus L. sect. Persica are not only important fruit trees, but also popular ornamental and medicinal plants. Correct identification of seedlings, barks, or fruit kernels is sometimes required, but no reliable morphological characters are available. Nowadays, the technique of DNA barcoding has the potential to meet such requirements. In this study, we evaluated the suitability of 11 DNA loci (atpB‐rbcL, trnH‐psbA, trnL‐F, trnS‐G, atpF‐H, rbcL, matK, rpoB, rpoC1, nad1, and internal transcribed spacer [ITS]) as candidate DNA barcodes for peaches, using samples from 38 populations, covering all the species in sect. Persica. On the whole, the primers worked well in this group and sequencing difficulties were met only in the case of ITS locus. Five loci (rbcL, matK, rpoB, rpoC, and nad1) have very low variation rates, whereas atpB‐rbcL, atpF‐H, trnH‐psbA, trnL‐F and trnS‐G show more variability. The most variable loci, atpB‐rbcL and trnH‐psbA, can distinguish three of the five species. Two two‐locus combinations, atpB‐rbcL+trnL‐F and atpB‐rbcL+atpF‐H, can resolve all five species. We also find that identification powers of the loci are method‐dependent. The NeighborNet method shows higher species identification power than maximum parsimony, neighbor joining, and unweighted pair group method with arithmetic mean methods.