Abstract Premise Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis. Methods FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana). Results We observed that FUSE can extract high quantities of DNA in 9–15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next‐generation sequencing. Discussion These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field‐deployable systems to increase the cataloging of genetic biodiversity, particularly in low‐resource biodiversity hotspots.

We evaluated an alternative small stem assay (AltSSA) for blight resistance in backcross hybrid chestnut trees (Castanea dentata/mollissima). Whereas standard small stem assays (SSAs) are done by inoculating small incisions in stems, in our AltSSA, 4- to 5-mm stems are cut off, and the exposed (living) stem tips are inoculated with discs of Cryphonectria parasitica inoculum and temporarily covered with plastic sleeves. Intended primarily for forward selection, this method was designed to be easy to implement, to consistently induce cankering, and to better enable seedling recovery via the development of lateral shoots from the lower stem. After 90+ days, cankers are evaluated and removed, and seedlings are prepared for out-planting. Previous results showed that AltSSAs performed at least as well as a common SSA method in distinguishing resistant and susceptible types. In this follow-up analysis of 35 lines of backcross seedlings studied in 2020 and 2021, we showed that mean orange zone canker length (OZCL) and a multifactor principal components analysis-based blight resistance index gave results consistent with predictions derived from two methods of blight resistance phenotyping and percentage of American chestnut ancestry of the parents of each line. As expected, based upon the apparent polygenic inheritance of blight resistance in backcross chestnut trees, mean OZCL of backcross families ranged from intermediate (F-1 hybrid-level) to low (wild-type American chestnut-level). Consistent with prior results, canker production was near 100%, survivorship after out-planting was very high, and postinoculation stem dieback was not apparently related to the stem tip inoculations. Altogether, these results suggest that the AltSSA is a viable method for early detection of relative blight resistance in seedlings and may enable a reduction in the numbers of trees out-planted and placed under care for long-term evaluation and breeding. Thus, the AltSSA can prevent time, resources, and orchard space from being used on susceptible trees.

Rapid DNA extraction is a critical barrier for routine and fieldable genetics tests for applications in conservation, such as illegal trafficking and fraudulent mislabelling. Here, we develop a non‐thermal focused ultrasound extraction (FUSE) technique that creates a dense cloud of high‐pressure acoustic cavitation bubbles to disintegrate targeted tissues into an acellular debris, resulting in the rapid release of entrapped DNA. In this work, we demonstrate the proof‐of‐concept of the FUSE technique by obtaining species identifiable sequences and shotgun sequencing reads from DNA extracted from Atlantic salmon Salmo salar tissues. Having mitigated the key risks for this technique, we hypothesize future developments with this technology can be applied to accelerate and simplify DNA extraction from exceedingly difficult samples with complex tissue matrices (i.e. fibrous tissue and timber samples) in both laboratory and field settings.

American chestnut was once a foundation species of eastern North American forests, but was rendered functionally extinct in the early 20th century by an exotic fungal blight ( Cryphonectria parasitica ). Over the past 30 years, The American Chestnut Foundation (TACF) has pursued backcross breeding to generate hybrids that combine the timber-type form of American chestnut with the blight tolerance of Chinese chestnut. The backcross strategy has been implemented based on the hypothesis that blight tolerance is conferred by few major effect alleles. We tested this hypothesis by developing genomic prediction models for five presence/absence blight phenotypes of 1,230 BC 3 F 2 selection candidates and average canker severity of their BC 3 F 3 progeny. We also genotyped pure Chinese and American chestnut reference panels to estimate the proportion of BC 3 F 2 genomes inherited from parent species. We found that genomic prediction from a method that assumes an infinitesimal model of inheritance (HBLUP) has a similar predictive ability to a method that tends to perform well for traits controlled by major genes (Bayes C). Furthermore, the proportion of BC 3 F 2 trees’ genomes inherited from American chestnut was negatively correlated with the blight tolerance of BC 3 F 2 trees and their progeny. On average, selected BC 3 F 2 trees inherited 83% of their genome from American chestnut and have blight-tolerance that is intermediate between F 1 hybrids and American chestnut. Results suggest polygenic rather than major gene inheritance for blight tolerance. The blight-tolerance of restoration populations will be enhanced by advancing additional sources of blight-tolerance through fewer backcross generations and by potentially by breeding with transgenic blight-tolerant trees.

American chestnut was once a foundation species of eastern North American forests, but was rendered functionally extinct in the early 20th century by an exotic fungal blight (Cryphonectria parasitica). Over the past 30 years, the American Chestnut Foundation (TACF) has pursued backcross breeding to generate hybrids that combine the timber-type form of American chestnut with the blight resistance of Chinese chestnut based on a hypothesis of major gene resistance. To accelerate selection within two backcross populations that descended from two Chinese chestnuts, we developed genomic prediction models for five presence/absence blight phenotypes of 1,230 BC3F2 selection candidates and average canker severity of their BC3F3 progeny. We also genotyped pure Chinese and American chestnut reference panels to estimate the proportion of BC3F2 genomes inherited from parent species. We found that genomic prediction from a method that assumes an infinitesimal model of inheritance (HBLUP) has similar accuracy to a method that tends to perform well for traits controlled by major genes (Bayes C). Furthermore, the proportion of BC3F2 trees' genomes inherited from American chestnut was negatively correlated with the blight resistance of these trees and their progeny. On average, selected BC3F2 trees inherited 83% of their genome from American chestnut and have blight resistance that is intermediate between F-1 hybrids and American chestnut. Results suggest polygenic inheritance of blight resistance. The blight resistance of restoration populations will be enhanced through recurrent selection, by advancing additional sources of resistance through fewer backcross generations, and by potentially by breeding with transgenic blight-tolerant trees.