Exploring the genomic basis of traits relevant to evolution and ecology of chestnuts (Castanea) using high-throughput DNA sequencing and bioinformatics.

Introduced pests and pathogens have devastated forest ecosystems in the temperate zone; in eastern North America, introduced pests and pathogens have led to the elimination of most mature elms (Ulmus), ashes (Fraxinus), hemlocks (Tsuga) and chestnuts (Castanea) over large areas where these genera were formerly abundant and important for local ecosystems. The restoration of species affected by introduced pests and pathogens requires the development and propagation of trees that possess heritable resistance. High-throughput DNA sequencing and genomics provide opportunities for researchers to identify resistance gene candidates, screen germplasm, and develop markers for marker-assisted selection in breeding programs, with the goal of restoring ecologically important wild trees to the landscape. American chestnut (Castanea dentata) is currently the focus of a major research effort that intends to restore the species by incorporating blight resistance from Chinese chestnut (Castanea mollissima), a species that is generally resistant to chestnut blight. I investigated several aspects of chestnut genomics and blight resistance with the goal of aiding the blight resistance breeding program for American chestnut. I tested a detached-leaf assay for chestnut blight resistance and learned that it may not be useful for screening advanced backcross (BC3) progeny in chestnut blight resistance breeding programs (Chapter 2). Utilizing a recent draft assembly of the Chinese chestnut reference genome, I analyzed patterns of genetic variation across regions associated with chestnut blight resistance, and found that several loci associated with blight resistance show markedly elevated nucleotide diversity in the most resistant Chinese chestnuts relative to more susceptible trees. At other blight-associated loci, genetic diversity was low in all C. mollissima (Chapter 3). This indicates that while maintaining high allelic diversity at blight resistance loci is desirable for a resistance breeding program, it may not be essential. Assessing potential unintended effects of hybrid breeding on the ecological behavior of restored chestnuts, I found that several genetic loci in third backcross (BC3) chestnut appear to affect caching decisions by squirrels due to inheritance of C. mollissima alleles that influence seed traits (Chapter 4). The reason for backcrossing in the American chestnut breeding program is to avoid the short, branchy mature form of C. mollissima. By sequencing the genomes of wild and orchard-derived Chinese chestnuts, I showed that some genomic loci under selection in orchard chestnuts (i.e., artificially selected by humans) may influence crown form (Chapter 5). This work should provide the basis for further investigations that validate the phenotypic effects of the proposed candidate genes, and utilize information on genetic polymorphisms identified here to accelerate chestnut improvement programs

The influence of genetic and environmental factors on morbidity and mortality in populations of butternut affected by butternut canker disease

Butternut (Juglans cinerea L.), or white walnut, has suffered large population declines in the past half-century due to poor regeneration and mortality caused by an introduced fungus, Ophiognomonia clavigignenti-juglandacearum (Nair, Kostichka & Kuntz) Broders & Boland. This fungus causes branch and trunk cankers that can coalesce to girdle adult trees. Chapter 1 provides background information on butternut and butternut canker. We used next-generation sequencing to identify new nuclear DNA markers for butternut and Japanese walnut, a congener with which butternut readily hybridizes. We also examined the alignment of SSR repeat sequences in butternut and Japanese walnut with similar sequences from other angiosperms in public sequence databases. The methods used and results obtained in this process are detailed in Chapter 2. Chapter 3 summarizes an investigation of the environmental and genetic factors contributing to canker disease incidence, severity, and mortality in a large (n=113) population of butternut in southern Wisconsin and two other populations of butternut, one near the main study site in southern Wisconsin and another in the Great Smoky Mountains National Park. We present evidence for weak correlations of genetic similarity and phenotypic similarity for several disease traits, parentage analysis of regeneration in the smaller Wisconsin population, and evidence for significant microsite influences on butternut mortality over an 11-year period in the large Slocum\u27s Woods butternut population

Exploring the Genomic Basis of Traits Relevant to Evolution and Ecology of Chestnut (Castanea) Using High-throughput DNA Sequencing and Bioinformatics

Introduced pests and pathogens have devastated forest ecosystems in the temperate zone; in eastern North America, introduced pests and pathogens have led to the elimination of most mature elms (Ulmus), ashes (Fraxinus), hemlocks (Tsuga) and chestnuts (Castanea) over large areas where these genera were formerly abundant and important for local ecosystems. The restoration of species affected by introduced pests and pathogens requires the development and propagation of trees that possess heritable resistance. High-throughput DNA sequencing and genomics provide opportunities for researchers to identify resistance gene candidates, screen germplasm, and develop markers for marker-assisted selection in breeding programs, with the goal of restoring ecologically important wild trees to the landscape. American chestnut (Castanea dentata) is currently the focus of a major research effort that intends to restore the species by incorporating blight resistance from Chinese chestnut (Castanea mollissima), a species that is generally resistant to chestnut blight. I investigated several aspects of chestnut genomics and blight resistance with the goal of aiding the blight resistance breeding program for American chestnut. I tested a detached-leaf assay for chestnut blight resistance and learned that it may not be useful for screening advanced backcross (BC3) progeny in chestnut blight resistance breeding programs (Chapter 2). Utilizing a recent draft assembly of the Chinese chestnut reference genome, I analyzed patterns of genetic variation across regions associated with chestnut blight resistance, and found that several loci associated with blight resistance show markedly elevated nucleotide diversity in the most resistant Chinese chestnuts relative to more susceptible trees. At other blight-associated loci, genetic diversity was low in all C. mollissima (Chapter 3). This indicates that while maintaining high allelic diversity at blight resistance loci is desirable for a resistance breeding program, it may not be essential. Assessing potential unintended effects of hybrid breeding on the ecological behavior of restored chestnuts, I found that several genetic loci in third backcross (BC3) chestnut appear to affect caching decisions by squirrels due to inheritance of C. mollissima alleles that influence seed traits (Chapter 4). The reason for backcrossing in the American chestnut breeding program is to avoid the short, branchy mature form of C. mollissima. By sequencing the genomes of wild and orchard-derived Chinese chestnuts, I showed that some genomic loci under selection in orchard chestnuts (i.e., artificially selected by humans) may influence crown form (Chapter 5). This work should provide the basis for further investigations that validate the phenotypic effects of the proposed candidate genes, and utilize information on genetic polymorphisms identified here to accelerate chestnut improvement programs

Signatures of Selection in the Genomes of Chinese Chestnut (Castanea mollissima Blume): The Roots of Nut Tree Domestication

Chestnuts (Castanea) are major nut crops in East Asia and southern Europe, and are unique among temperate nut crops in that the harvested seeds are starchy rather than oily. Chestnut species have been cultivated for three millennia or more in China, so it is likely that artificial selection has affected the genome of orchard-grown chestnuts. The genetics of Chinese chestnut (Castanea mollissima Blume) domestication are also of interest to breeders of hybrid American chestnut, especially if the low-growing, branching habit of Chinese chestnut, an impediment to American chestnut restoration, is partly the result of artificial selection. We resequenced genomes of wild and orchard-derived Chinese chestnuts and identified selective sweeps based on pooled whole-genome SNP datasets. We present candidate gene loci for chestnut domestication and discuss the potential phenotypic effects of candidate loci, some of which may be useful genes for chestnut improvement in Asia and North America. Selective sweeps included predicted genes potentially related to flower phenology and development, fruit maturation, and secondary metabolism, and included some genes homologous to domestication candidates in other woody plants

Data_Sheet_1_Signatures of Selection in the Genomes of Chinese Chestnut (Castanea mollissima Blume): The Roots of Nut Tree Domestication.XLSX

<p>Chestnuts (Castanea) are major nut crops in East Asia and southern Europe, and are unique among temperate nut crops in that the harvested seeds are starchy rather than oily. Chestnut species have been cultivated for three millennia or more in China, so it is likely that artificial selection has affected the genome of orchard-grown chestnuts. The genetics of Chinese chestnut (Castanea mollissima Blume) domestication are also of interest to breeders of hybrid American chestnut, especially if the low-growing, branching habit of Chinese chestnut, an impediment to American chestnut restoration, is partly the result of artificial selection. We resequenced genomes of wild and orchard-derived Chinese chestnuts and identified selective sweeps based on pooled whole-genome SNP datasets. We present candidate gene loci for chestnut domestication and discuss the potential phenotypic effects of candidate loci, some of which may be useful genes for chestnut improvement in Asia and North America. Selective sweeps included predicted genes potentially related to flower phenology and development, fruit maturation, and secondary metabolism, and included some genes homologous to domestication candidates in other woody plants.</p

Image_1_Signatures of Selection in the Genomes of Chinese Chestnut (Castanea mollissima Blume): The Roots of Nut Tree Domestication.pdf

<p>Chestnuts (Castanea) are major nut crops in East Asia and southern Europe, and are unique among temperate nut crops in that the harvested seeds are starchy rather than oily. Chestnut species have been cultivated for three millennia or more in China, so it is likely that artificial selection has affected the genome of orchard-grown chestnuts. The genetics of Chinese chestnut (Castanea mollissima Blume) domestication are also of interest to breeders of hybrid American chestnut, especially if the low-growing, branching habit of Chinese chestnut, an impediment to American chestnut restoration, is partly the result of artificial selection. We resequenced genomes of wild and orchard-derived Chinese chestnuts and identified selective sweeps based on pooled whole-genome SNP datasets. We present candidate gene loci for chestnut domestication and discuss the potential phenotypic effects of candidate loci, some of which may be useful genes for chestnut improvement in Asia and North America. Selective sweeps included predicted genes potentially related to flower phenology and development, fruit maturation, and secondary metabolism, and included some genes homologous to domestication candidates in other woody plants.</p

Training Population Optimization for Genomic Selection in Miscanthus

Miscanthus is a perennial grass with potential for lignocellulosic ethanol production. To ensure its utility for this purpose, breeding efforts should focus on increasing genetic diversity of the nothospecies Miscanthus x giganteus (M·g) beyond the single clone used in many programs. Germplasm from the corresponding parental speciesM. sinensis (Msi) and M. sacchariflorus (Msa) could theoretically be used as training sets for genomic prediction of M·g clones with optimal genomic estimated breeding values for biofuel traits. To this end, we first showed that subpopulation structure makes a substantial contribution to the genomic selection (GS) prediction accuracies within a 538-member diversity panel of predominately Msi individuals and a 598-member diversity panels of Msa individuals. We then assessed the ability of these two diversity panels to train GS models that predict breeding values in an interspecific diploid 216-member M·g F2 panel. Low and negative prediction accuracies were observed when various subsets of the two diversity panels were used to train theseGSmodels. To overcome the drawback of having only one interspecific M·g F2 panel available, we also evaluated prediction accuracies for traits simulated in 50 simulated interspecific M·g F2 panels derived from different sets of Msi and diploidMsa parents. The results revealed that genetic architectures with common causalmutations across Msi and Msa yielded the highest prediction accuracies. Ultimately, these results suggest that the ideal training set should contain the same causal mutations segregating within interspecific M·g populations, and thus efforts should be undertaken to ensure that individuals in the training and validation sets are as closely related as possible

The leaf economics spectrum of triploid and tetraploid C4 grass Miscanthus x giganteus.

Funder: DE‐SC0018420Funder: U.S. Department of Energy; Id: http://dx.doi.org/10.13039/100000015The leaf economics spectrum (LES) describes multivariate correlations in leaf structural, physiological and chemical traits, originally based on diverse C3 species grown under natural ecosystems. However, the specific contribution of C4 species to the global LES is studied less widely. C4 species have a CO2 concentrating mechanism which drives high rates of photosynthesis and improves resource use efficiency, thus potentially pushing them towards the edge of the LES. Here, we measured foliage morphology, structure, photosynthesis, and nutrient content for hundreds of genotypes of the C4 grass Miscanthus× giganteus grown in two common gardens over two seasons. We show substantial trait variations across M.× giganteus genotypes and robust genotypic trait relationships. Compared to the global LES, M.× giganteus genotypes had higher photosynthetic rates, lower stomatal conductance, and less nitrogen content, indicating greater water and photosynthetic nitrogen use efficiency in the C4 species. Additionally, tetraploid genotypes produced thicker leaves with greater leaf mass per area and lower leaf density than triploid genotypes. By expanding the LES relationships across C3 species to include C4 crops, these findings highlight that M.× giganteus occupies the boundary of the global LES and suggest the potential for ploidy to alter LES traits

Training Population Optimization for Genomic Selection in Miscanthus

Miscanthus is a perennial grass with potential for lignocellulosic ethanol production. To ensure its utility for this purpose, breeding efforts should focus on increasing genetic diversity of the nothospecies Miscanthus x giganteus (M·g) beyond the single clone used in many programs. Germplasm from the corresponding parental speciesM. sinensis (Msi) and M. sacchariflorus (Msa) could theoretically be used as training sets for genomic prediction of M·g clones with optimal genomic estimated breeding values for biofuel traits. To this end, we first showed that subpopulation structure makes a substantial contribution to the genomic selection (GS) prediction accuracies within a 538-member diversity panel of predominately Msi individuals and a 598-member diversity panels of Msa individuals. We then assessed the ability of these two diversity panels to train GS models that predict breeding values in an interspecific diploid 216-member M·g F2 panel. Low and negative prediction accuracies were observed when various subsets of the two diversity panels were used to train theseGSmodels. To overcome the drawback of having only one interspecific M·g F2 panel available, we also evaluated prediction accuracies for traits simulated in 50 simulated interspecific M·g F2 panels derived from different sets of Msi and diploidMsa parents. The results revealed that genetic architectures with common causalmutations across Msi and Msa yielded the highest prediction accuracies. Ultimately, these results suggest that the ideal training set should contain the same causal mutations segregating within interspecific M·g populations, and thus efforts should be undertaken to ensure that individuals in the training and validation sets are as closely related as possible