Development of a quantitative PCR assay for detection of redside shiner (Richardsonius balteatus) from environmental DNA.

OBJECTIVE: A quantitative PCR (qPCR) assay for the detection of redside shiner (Richardsonius balteatus) environmental DNA (eDNA) was designed as a side product of a larger project aimed at using eDNA to determine the presence and geographic extent of native and non-native fishes in the reservoirs and associated tributaries above the three mainstem dams (Ross, Diablo, Gorge) on the Skagit River, Washington, USA. The eDNA survey results can be used to help guide additional sampling efforts that include traditional sampling methods, such as electrofishing and netting. RESULTS: The redside shiner qPCR assay (RSSCOI_540-601) was validated by testing for sensitivity using redside shiner genomic DNA from three different populations and by testing for specificity against 30 potentially sympatric species. No non-target amplification was observed in our validation tests. We then evaluated the assay on field-collected water samples where there are known populations of redside shiner and a negative control site where the target species is known to be absent. The field-collected water samples tested positive at the redside shiner sites and tested negative at the negative control site. The assay could provide resource managers with an effective means for surveying and monitoring redside shiner populations.

Development of a molecular diagnostic system to discriminate Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel).

A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.

Stress tolerance in plants via habitat-adapted symbiosis.

We demonstrate that native grass species from coastal and geothermal habitats require symbiotic fungal endophytes for salt and heat tolerance, respectively. Symbiotically conferred stress tolerance is a habitat-specific phenomenon with geothermal endophytes conferring heat but not salt tolerance, and coastal endophytes conferring salt but not heat tolerance. The same fungal species isolated from plants in habitats devoid of salt or heat stress did not confer these stress tolerances. Moreover, fungal endophytes from agricultural crops conferred disease resistance and not salt or heat tolerance. We define habitat-specific, symbiotically-conferred stress tolerance as habitat-adapted symbiosis and hypothesize that it is responsible for the establishment of plants in high-stress habitats. The agricultural, coastal and geothermal plant endophytes also colonized tomato (a model eudicot) and conferred disease, salt and heat tolerance, respectively. In addition, the coastal plant endophyte colonized rice (a model monocot) and conferred salt tolerance. These endophytes have a broad host range encompassing both monocots and eudicots. Interestingly, the endophytes also conferred drought tolerance to plants regardless of the habitat of origin. Abiotic stress tolerance correlated either with a decrease in water consumption or reactive oxygen sensitivity/generation but not to increased osmolyte production. The ability of fungal endophytes to confer stress tolerance to plants may provide a novel strategy for mitigating the impacts of global climate change on agricultural and native plant communities.