We are using the CRISPR-Cas9 genome editing system to test for adaptation of two genes with putative roles in climatic stress in sugar pine trees. Understanding the genetic basis of adaptive traits has been central to ecology and evolution ever since the Darwinian revolution. Modern landscape genomics has made advances in identifying genes that are associated with phenotypic expression, but they have been unable to prove that the associations are more than correlative. The threats to biological diversity raised by climate change underscore the need to have an improved understanding of the genetic basis of phenotypic traits. In sedentary, long-lived tree species this becomes of utmost importance, as the success of populations is likely to depend, in large part, on existing genetic variation.

We have selected the CaPF1 and WRKY genes that are known to play a role in drought and temperature stress, and will first document their variation across an environmental gradient in the western conifer, sugar pine (Pinus lambertiana). The annotated genome of sugar pine was recently published and will help us in our study. Four populations will be sampled that are representative of climatic breaks in the range of the species. We will test the function of the allelic variants for their role in drought and temperature tolerance using the CRISPR-Cas9 genome editing system. This will be the first time that genome-editing tools have been used in studies of gymnosperms. Pine embryos will be used to provide the source of material for tissue culture and subsequent transformation. NHEJ repair is expected to provide the knockout mutation for testing the role of the genes that will be evaluated through drought and temperature stress tests. Polyamines are believed to mediate the CaPF1 response and so levels of polyamines will be determined by HPLC.

Once the role of gene action has been determined, we will attempt to transform tissue from the different populations by knock-ins of the different alleles. This will require the HDR pathway. By conducting reciprocal allele transfers among populations, we will be able to evaluate landscape levels of adaptation to climatic stresses. Our research will provide an important and novel use of the CRISPR-Cas9 technology in its application to studies of ecology and evolution, to long-lived plants and, in particular, conifers.