On the origin and dispersal of cultivated spinach (Spinacia oleracea L.)

Spinach (Spinacia oleracea L.) is an economically important crop that is cultivated and consumed worldwide. Spinach is interfertile with the wild species S. tetrandra Steven ex M. Bieb. and S. turkestanica Iljin that therefore are presumed to include the most likely crop ancestor. Here we studied variation in 60 Single Nucleotide Polymorphisms (SNP) previously identified in S. oleracea to address the issue of crop ancestry and domestication region. For this purpose we investigated 95 accessions, including 54 spinach landraces from a wide geographic area in Europe and Asia and 16 S. tetrandra and 25 S. turkestanica populations of which the majority had only recently become available. Compared to S. tetrandra substantially higher levels of amplification success and higher levels of variation were detected for S. turkestanica, indicating that S. oleracea is genetically closer to S. turkestanica than to S. tetrandra. Our phylogenetic and population structure analysis supported the conclusion that S. turkestanica is the most likely ancestor of cultivated spinach. In addition, these analyses revealed a group of S. oleracea landraces from Eastern and Southern Asia with a strong genetic resemblance to S. turkestanica. This group includes landraces from Afghanistan and Pakistan, which are part of the native distribution range of S. turkestanica. The domestication of spinach may therefore have occurred more eastwards than generally assumed. Furthermore, our study provides support for the hypothesis that after domestication, spinach was introduced into China via Nepal. Additional collecting of spinach landraces is recommended in order to allow the more precise reconstruction of the crop migration routes.

PCR primers comparisons for a successful Tuber spp. DNA region amplification in routine identifications / Primerjava PCR začetnih oligonukleotidov za uspešno pomnoževanje DNA regije Tuber spp. pri rutinski identifikaciji

Since late 20th century DNA sequencing became the method of choice method in precision species identification. The ITS region is one of the official fungal barcoding DNA markers, although in some cases sequencing of the ITS region may, due to misidentification, mislabeling or nomenclature errors in public databases, lead to incorrect or insufficient identification, as is currently a case in the genus Tuber. The aim of this study was to test, which ITS primer pairs are most appropriate and optimal for Tuber species DNA region amplification. Thereby we (1) compared amplification success for different Tuber species using fungal specific primer pair ITS1f and ITS4 and (2) compared amplification success using different ITS primer pair combinations in amplifying DNA region an example species Tuber aestivum. Based on results, Tuber aestivum was one of the most reluctant Tuber species in this study and in most cases failed to amplify with the above primer pair. After comparing different ITS primer pairs, we conclude that the primer pair ITS5 and ITS7 is the most appropriate primer pair for amplification DNA region of T. aestivum as it resulted in high amplification success from ectomycorrhizal root tips. Based on sequences, gained from public databases, we found that ITS1f and ITS6 primers have a mismatch in one base pair compared to the target sequence of Tuber aestivum, thus resulting in poor or no amplification success. Although primer pair ITS5 and ITS7 in our study was proven to be the most appropriate primer pair in amplifying DNA region Tuber aestivum species, further analysis about appropriateness of it for a general barcoding and identification of ectomycorrhiza in complex community samples is needed. Keywords: Tuber spp., ITS region, PCR amplification, ITS primers Izvleček Od konca 20. stoletja je določanje nukleotidnega zaporedja DNA postalo ena izmed pogosteje uporabljenih metod za določanje vrst. ITS regija je edna izmed uradnih glivnih DNA markerjev, čeprav lahko določanje nukleotidnega zaporedja le-te, v nekaterih primerih, predvsem zaradi napačne določitve, označevanja oziroma napak v nomenklaturi v javnih bazah podatkov, privede do napačne oziroma nenatančne določitve vrst, kar je trenutno težava pri določitvi vrst iz rodu Tuber. Namen te študije je bil testirati kateri pari ITS začetnih oligonukleotidov so najbolj primerni in optimalni za pomnoževanje DNA regij gliv iz rodu Tuber. S tem namenom smo v študiji (1) primerjali uspešnost pomnoževanja DNA regije različnih vrst iz rodu Tuber, z uporabo glivno specifičnih začetnih oligonukleotidov ITS1f in ITS4 ter hkrati (2) primerjali uspešnost pomnoževanja DNA regije vrste Tuber aestivum z uporabo različnih ITS začetnih oligonukleotidov. Na podlagi rezultatov ugotavljamo, da je vrsta T. aestivum izmed vseh analiziranih gliv iz rodu Tuber, bila najtežavnejša vrsta v naši študiji, saj je v večini primerov pomnoževanje DNA regije te vrste z uporabo glivno specifičnih začetnih oligonukleotidov ITS1f in ITS4 bilo neuspešno. Po primerjavi uspešnosti pomnoževanja z različnimi ITS začetnimi oligonukelotidi ugotavljamo, da sta bila v naši študiji ITS začetna oligonukleotida ITS5 in ITS7 najprimernejša za pomnoževanje DNA regije vrste T. aestivum, saj je bila uspešnost pomnoževanja iz ektomikoriznih vršičkov v tem primeru največja. Na podlagi T. aestivum nukleotidnih zaporedij pridobljenih iz javnih podatkovnih baz ugotavljamo, da je za začetna oligonukleotida ITS1f in ITS6 značilno neujemanje s tarčnim nukleotidnim zaporedjem (T. aestivum) v enem baznem paru, kar se lahko odraža bodisi v slabšem pomnoževalnem uspehu ali v nepomnoževanju na splošno. Kljub temu, da v naši študiji ugotavljamo, da sta začetna oligonukleotida ITS5 in ITS7 najprimernejša za pomnoževanje DNA regije glive T. aestivum, so potrebne nadaljnje analize, s katerimi bi potrdili splošno primernost omenjenega para ITS5/ITS7 za pomnoževanje DNA regije ne samo vrst iz rodu Tuber, temveč za določanje ektomikoriznih glivnih združb na splošno. Ključne besede: Tuber spp., ITS regija, PCR pomno­ževanje, ITS začetni oligonukleotidi

Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants

Objective The objective of this study is to develop and identify polymorphic microsatellite markers for fungus-gardening (attine) ants in the genus Trachymyrmex sensu lato . These ants are important ecosystem engineers and have been a model group for understanding complex symbiotic systems, but very little is understood about the intraspecific genetic patterns across most North American attine species. These microsatellite markers will help to better study intraspecific population genetic structure, gene flow, mating habits, and phylogeographic patterns in these species and potentially other congeners. Results Using next-generation sequencing techniques, we identified 17 and 12 polymorphic microsatellite markers from T. septentrionalis and Mycetomoellerius (formerly Trachymyrmex ) turrifex , respectively, and assessed the genetic diversity of each marker. We also analyzed the cross-amplification success of the T. septentrionalis markers in two other closely related Trachymyrmex species, and identified 10 and 12 polymorphic markers for T. arizonensis and T. pomonae , respectively.

Development, characterization, and cross-amplification of polymorphic microsatellite markers for North American Trachymyrmex and Mycetomoellerius ants

Objective The objective of this study is to develop and identify polymorphic microsatellite markers for fungus-gardening (attine) ants in the genus Trachymyrmex sensu lato . These ants are important ecosystem engineers and have been a model group for understanding complex symbiotic systems, but very little is understood about the intraspecific genetic patterns across most North American attine species. These microsatellite markers will help to better study intraspecific population genetic structure, gene flow, mating habits, and phylogeographic patterns in these species and potentially other congeners. Results Using next-generation sequencing techniques, we identified 17 and 12 polymorphic microsatellite loci from T. septentrionalis and Mycetomoellerius (formerly Trachymyrmex ) turrifex , respectively, and assessed the genetic diversity of each locus. We also analyzed the cross-amplification success of the T. septentrionalis markers in two other closely related Trachymyrmex species, and identified 10 and 12 polymorphic markers for T. arizonensis and T. pomonae , respectively.

A novel method to optimise the utility of underused moulted plumulaceous feather samples for genetic analysis in bird conservation

Non-invasive sampling methods are increasingly being used in conservation research as they reduce or eliminate the stress and disturbance resulting from invasive sampling of blood or tissue. Here we present a protocol optimised for obtaining usable genetic material from moulted plumulaceous feather samples. The combination of simple alterations to a ‘user-developed’ method, comprised of increased incubation time and modification of temperature and volume of DNA elution buffer, are outlined to increase DNA yield and significantly increase DNA concentration (W = 81, p < 0.01, Cohens’s d = 0.89). We also demonstrate that the use of a primerless polymerase chain reaction (PCR) technique increases DNA quality and amplification success when used prior to PCR reactions targeting avian mitochondrial DNA (mtDNA). A small amplicon strategy proved effective for mtDNA amplification using PCR, targeting three overlapping 314–359 bp regions of the cytochrome oxidase I barcoding region which, when combined, aligned with target-species reference sequences. We provide evidence that samples collected non-invasively in the field and kept in non-optimal conditions for DNA extraction can be used effectively to sequence a 650 bp region of mtDNA for genetic analysis.

Consideration of sample source for establishing reliable genetic microsatellite data from mammalian carnivore specimens held in natural history collections

Specimens from natural history collections (NHCs) are increasingly being used for genetic studies and can provide information on extinct populations, facilitate comparisons of historical and contemporary populations, produce baseline data before environmental changes, and elucidate patterns of change. Destructive sampling for DNA may be in disagreement with NHC goals of long-term care and maintenance. Differentiating quality among sample sources can direct destructive sampling to the source predicted to yield the highest quality DNA and most reliable data, potentially reducing damage to specimens, laboratory costs, and genotyping errors. We used the kit fox (Vulpes macrotis) as a model species and evaluated the quality and reliability of genetic data obtained from carnivoran specimens via three different sample sources: cranial bones, nasal bones, and toepads. We quantified variation in microsatellite amplification success and genotyping error rates and assessed the reliability of source-specific genic data. Toepads had the highest amplification success rates and lowest genotyping error rates. Shorter loci had higher amplification success and lower allelic dropout rates than longer loci. There were substantial differences in the reliability of resulting multilocus genotypes. Toepads produced the most reliable data, required the fewest replicates, and therefore, had the lowest costs to achieve reliable data. Our results demonstrate that the quality of DNA obtained from specimens varies by sample source and can inform NHCs when evaluating requests for destructive sampling. Our results suggest that prior to large-scale specimen sampling, researchers should conduct pilot studies to differentiate among source-specific data reliability, identify high performing loci, reduce costs of analyses, and minimize destructive sampling.

Applying and refining DNA analysis to determine the identity of plant material extracted from the digestive tracts of katydids

Background Feeding habits are central to animal ecology, but it is often difficult to characterize the diet of organisms that are arboreal, nocturnal, rare, or highly mobile. Genetic analysis of gut contents is a promising approach for expanding our understanding of animal feeding habits. Here, we adapt a laboratory protocol for extracting and sequencing plant material from gut contents and apply it to Neotropical forest katydids (Orthoptera: Tettigoniidae) on Barro Colorado Island (BCI) in Panama. Methods Our approach uses three chloroplast primer sets that were previously developed to identify vegetation on BCI. We describe the utility and success rate of each primer set. We then test whether there is a significant difference in the amplification and sequencing success of gut contents based on the size or sex of the katydid, the time of day that it was caught, and the color of the extracted gut contents. Results We find that there is a significant difference in sequencing success as a function of gut color. When extracts were yellow, green, or colorless the likelihood of successfully amplifying DNA ranged from ~30–60%. When gut extracts were red, orange, or brown, amplification success was exceptionally low (0–8%). Amplification success was also higher for smaller katydids and tended to be more successful in katydids that were captured earlier in the night. Strength of the amplified product was indicative of the likelihood of sequencing success, with strong bands having a high likelihood of success. By anticipating which samples are most likely to succeed, we provide information useful for estimating the number of katydids that need to be collected and minimizing the costs of purifying, amplifying, and sequencing samples that are unlikely to succeed. This approach makes it possible to understand the herbivory patterns of these trophically important katydids and can be applied more broadly to understand the diet of other tropical herbivores.

Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method’s application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the “non-amplifiable” samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Testing skin swabbing for DNA sampling in dendrobatid frogs

Skin swabbing, a minimally invasive DNA sampling method recently proposed for adult amphibians, was tested on the dendrobatid frog Allobates femoralis. I compared DNA yield from skin swabs and toe clips by evaluating obtained DNA concentrations and purity of extracts, as well as amplification success using eleven polymorphic microsatellite loci. I also tested whether storing skin swabs for two months at −20°C affected the properties of the extract or microsatellite analysis. Results show that skin swabs of adult A. femoralis suffered from high contamination and yielded significantly lower DNA quality and quantity, resulting in insufficient genotyping success, than DNA obtained from toe clips. The relatively dry skin in dendrobatid frogs may have impeded the collection of sufficient viable cells, and the presence of skin alkaloids and microbiota in the frog mucus may lead to high contamination load of skin swabs.