Detection of seed DNA in regurgitates of granivorous carabid beetles

Granivory can play a pivotal role in influencing regeneration, colonization as well as abundance and distribution of plants. Due to their high abundance, nutrient content and longevity, seeds are an important food source for many animals. Among insects, carabid beetles consume substantial numbers of seeds and are thought to be responsible for a significant amount of seed loss. However, the processes that govern which seeds are eaten and are therefore prevented from entering the seedbank are poorly understood. Here, we assess if DNA-based diet analysis allows tracking the consumption of seeds by carabids. Adult individuals ofHarpalus rufipeswere fed with seeds ofTaraxacum officinaleandLolium perenneallowing them to digest for up to 3 days. Regurgitates were tested for the DNA of ingested seeds at eight different time points post-feeding using general and species-specific plant primers. The detection of seed DNA decreased with digestion time for both seed species, albeit in a species-specific manner. Significant differences in overall DNA detection rates were found with the general plant primers but not with the species-specific primers. This can have implications for the interpretation of trophic data derived from next-generation sequencing, which is based on the application of general primers. Our findings demonstrate that seed predation by carabids can be tracked, molecularly, on a species-specific level, providing a new way to unravel the mechanisms underlying in-field diet choice in granivores.

Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer–probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

Detection of Sesame Seed DNA in Foods Using Real-Time PCR

The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.

The utility of aged seeds in DNA banks

The long-term utility of DNA banks is predicated on the stability of DNA during storage. The quality and yield of DNA extracted from seeds from four garden species, which varied in age from 1 to 135 years old, was used to examine the early stages of DNA degradation. Seeds that were 70 years old yielded high molecular weight DNA, which permitted amplification of a 650 bp segment of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and a 487 bp segment of the plastid-encoded large subunit of rubisco (rbcL). DNA extracted from seeds estimated to be about 135 years old was degraded, but still permitted amplification when routine extraction procedures were used. The rbcL sequences obtained from c. 135-year-old cucurbit seed DNA were consistent with species identifications based on seed morphological features; however, ITS sequences from the same samples were determined to be of fungal origin. A comparison of our results with studies of DNA stability in leaf specimens suggested that DNA degraded within both tissues, but perhaps more slowly within seeds. Evidence that high-quality DNA can be extracted from old, non-viable seeds expands the utility of seed banks in preserving genetic resources.

Self-incompatibility of `Arbequina' and `Picual' Olive Assessed by SSR Markers

We studied the self-incompatibility of two main Spanish olive (Olea europaea L.) cultivars, `Picual' and `Arbequina', by testing the selfing of the seeds with microsatellites. For this purpose, we used a rapid single-seed DNA extraction method and four highly polymorphic microsatellites. We analyzed seeds produced in branches bagged for selfing from mono- and multi-cultivar orchards in 2002 and 2003. We did not find any seed coming from selfing in the bagged branches, for either cultivar, in the two types of orchards. Additionally, we tested seeds coming from free pollination in mono-cultivar orchards from different locations. In the case of `Picual' olive, only three seeds out of the 70 collected were the product of selfing, although they came from mono-cultivar orchards located in areas where the cultivar used as the female parent was predominant. From the 20 seeds of `Arbequina' olive harvested in the middle of two high-density plantations, not one was a product of selfing. According to this, olive would behave as an allogamous species in mono-cultivar growing conditions and the pollen coming from long distances would be able to produce a normal bearing. Therefore, there is strong evidence to support the idea that the cultivars studied could be self-incompatible. Future experiments in self-compatibility should include a paternity check of the possible self seeds obtained.

Environmentally induced polymorphisms detected by RAPD analysis of soybean seed DNA

Experiments were performed to determine if RAPD profiles developed using total DNA isolated from soybean seed could be affected by the physiological state or the quality of the seed. RAPD profiles were developed using template DNA isolated from a single seed lot of soybean (Glycine max L. cv. Kirby). High quality seeds were used to produce four populations varying in either quality or physiological state: untreated control seed ambient temperature and humidity storage for 12 months, accelerated aging at 41°C and 100% relative humidity for 48 h, and controlled hydration (seed priming). One hundred and eighty-eight primers were used to create separate RAPD profiles from total DNA isolated from each set of seed and from soybean leaf tissue. Sixteen polymorphisms from 14 primers were identified as a result of seed treatments. Six primers showed nine polymorphisms in RAPD profiles of ambient-stored seed DNA, while four and two primers produced polymorphisms in reactions using accelerated aged or primed-seed template DNA, respectively. Two primers showed a polymorphic fragment in vegetative DNA not observed in any of the seed DNA samples. Ten of the observed polymorphisms were due to the appearance of a DNA fragment in response to a specific seed treatment while six were the result of the treatment-induced loss of a DNA fragment. The six polymorphisms resulting from the loss of a major fragment were all due to ambient-temperature seed storage. Results were reproducibly obtained from multiple DNA isolations using three separate DNA isolation procedures involving either multiple seed or a single seed as the template source. Therefore, genetically identical seed can consistently display RAPD polymorphisms as a response to the environmental exposure.