Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory

Data from: How generalist herbivores exploit belowground plant diversity in temperate grasslands

Belowground herbivores impact plant performance, thereby inducing changes in plant community composition, which potentially leads to cascading effects onto higher trophic levels and ecosystem processes and productivity. Amongst soil-living insects, external root-chewing generalist herbivores have the strongest impact on plants. However, the lack of knowledge on their feeding behaviour under field conditions considerably hampers achieving a comprehensive understanding of how they affect plant communities. Here, we address this gap of knowledge by investigating the feeding behaviour of Agriotes click beetle larvae, which are common generalist external root-chewers in temperate grassland soils. Utilizing diagnostic multiplex PCR to assess the larval diet, we examined the seasonal patterns in feeding activity, putative preferences for specific plant taxa, and whether species identity and larval instar affects food choices of the herbivores. Contrary to our hypothesis, most of the larvae were feeding-active throughout the entire vegetation period, indicating that the grassland plants are subjected to constant belowground feeding pressure. Feeding was selective, with members of Plantaginaceae and Asteraceae being preferred; Apiaceae were avoided. Poaceae, although assumed to be most preferred, had an intermediate position. The food preferences exhibited seasonal changes, indicating a fluctuation in plant traits important for wireworm feeding choice. Species- and instar-specific differences in dietary choice of the Agriotes larvae were small, suggesting that species and larval instars occupy the same trophic niche. According to the current findings, the food choice of these larvae is primarily driven by plant identity, exhibiting seasonal changes. This needs to be considered when analysing soil herbivore-plant interactions

Plant species list used for determining sensitivity of multiplex (FLPS, LF, TAT, TZ) and singleplex assays (Plantago, Apiaceae, Poaceae).

<p>Lowest detection rates achieved are given in number of template copies.</p

Plant species collected in maize fields (M) and perennial grassland (G), which were used to establish the PCR-based identification system.

<p>Target species of the molecular assays are displayed in bold.</p

Relative location of primer binding sites on the <i>trn</i>T-F cpDNA region.

<p>At the base of each figure is a size marker, which indicates a sequence length of 50 bp. (<b>A</b>) Positions of the family specific primers for Poaceae (above) and Apiaceae (below): The dotted lines represent the known sequence, the dashed lines the second exon of <i>trn</i>L and the exon of <i>trn</i>F, and the thick bars symbolise the primer binding sites. (<b>B</b>) Position of the genus- and species specific primers: The dotted lines represent the known sequence, the inner bars indicate the position of the two <i>trn</i>L exons and the outer bars the position of the <i>trn</i>T and the <i>trn</i>F gen. The binding sites of primers are indicated by double crosses.</p