Keith E. Woeste

Technological advances in temperate hardwood tree improvement including breeding and molecular marker applications

Hardwood forests and plantations are an important economic resource for the forest products industry worldwide and to the international trade of lumber and logs. Hardwood trees are also planted for ecological reasons, for example, wildlife habitat, native woodland restoration, and riparian buffers. The demand for quality hardwood from tree plantations will continue to rise as the worldwide consumption of forest products increases. Tree improvement of temperate hardwoods has lagged behind that of coniferous species and hardwoods of the genera Populus and Eucalyptus. The development of marker systems has become an almost necessary complement to the classical breeding and improvement of hardwood tree populations for superior growth, form, and timber characteristics. Molecular markers are especially valuable for determining the reproductive biology and population structure of natural forests and plantations, and the identity of genes affecting quantitative traits. Clonal reproduction of commercially important hardwood tree species provides improved planting stock for use in progeny testing and production forestry. Development of in vitro and conventional vegetative propagation methods allows mass production of clones of mature, elite genotypes or genetically improved genotypes. Genetic modification of hardwood tree species could potentially produce trees with herbicide tolerance, disease and pest resistance, improved wood quality, and reproductive manipulations for commercial plantations. This review concentrates on recent advances in conventional breeding and selection, molecular marker application, in vitro culture, and genetic transformation, and discusses the future challenges and opportunities for valuable temperate (or “fine”) hardwood tree improvement.

A robust set of black walnut microsatellites for parentage and clonal identification

We describe the development of a robust and powerful suite of 12 microsatellite marker loci for use in genetic investigations of black walnut and related species. These 12 loci were chosen from a set of 17 candidate loci used to genotype 222 trees sampled from a 38-year-old black walnut progeny test. The 222 genotypes represent a sampling from the broad geographic distribution of the species. Analysis of the samples using the 12 loci revealed an average expected heterozygosity of 0.83, a combined probability of identity of 3×10−19, and a combined probability of exclusion for paternity analysis of >0.999. The 222 genotyped trees from the progeny test comprised 39 open-pollinated families, 29 of which (having at least five sampled progeny) were used to estimate the outcrossing rate for the progeny trial. The same 29 families were used to construct a Neighbor-Joining dendrogram based upon allele sharing between individuals. The multilocus estimate of the outcrossing rate was 100% (standard error of zero), higher than the 90% level found in previous studies at the embryo stage, suggesting that both artificial and natural selection against selfs may have occurred over the 38-year lifespan of the progeny trial. In the Neighbor-Joining dendrogram, the majority of the putative siblings grouped together in 21 out of the 29 families, showing that the microsatellites were able to discern most of the family structure in the dataset. Our results indicate that errors were sometimes committed during the establishment of the progeny test. This set of microsatellite loci clearly provides a powerful tool for future applications in black walnut.

Using molecular methods to improve the genetic management of captive breeding programs for threatened species

Captive breeding programs are powerful tools for the conservation of our natural resources. Both animal and plant biodiversity are in global decline (International Union for Conservation of Nature [IUCN] 2008), and the trend is for levels and rates of endangerment to continue increasing (Butchart et al. 2005; Chapter 1 by Honeycutt and colleagues). When in-situ conservation efforts are insufficient at stopping or reversing the decline of a species, captive populations often represent the only alternative for forestalling extinction. For example, it is currently estimated that hundreds of amphibian species will soon be extinct if emergency measures are not taken to protect populations in captivity until the threats to wild populations can be halted or overcome (Gascon et al. 2007). The success of numerous captive breeding programs has been well documented. As just a few of many examples, captive breeding programs have saved the black-footed ferret (Mustela nigripes), Przewalski’s horse (Equus caballus przewalskii), and the California condor (Gymnogyps californianus) from final extinction (after the last wild populations were extirpated) and new wild populations of the golden lion tamarin (Leontopithecus rosalia), Arabian oryx (Oryx leucoryx), and whooping crane (Grus americana) have been successfully re-established from captive stocks. Conservation breeding programs aim to maintain populations that are representative of their wild counterparts, to provide a reservoir for future reintroductions and recovery efforts (see Chapter 12 by Rhodes and Latch). Thus, the genetic goals of captive population management are to minimize genetic drift, retain genetic diversity, restrict inbreeding, and limit adaptation to captivity (Lacy 1994). The foundations of most captive breeding programs are pedigree analyses, which are used to manage both the demography and genetics of captive populations (Ballou & Foose 1996). Accurate pedigrees provide information on inbreeding, the kinships among individuals, and the distributions of individual founder contributions to a population. Collectively, this information is used to produce regular breeding recommendations intended to meet demographic and genetic goals. Although pedigree analyses are quite effective, they do have limitations. The most basic limitation of pedigree-based management is that it requires complete and accurate pedigrees to be effective. Thus, the genetic and demographic management of captive populations are hampered when missing or inaccurate parentage records produce incomplete pedigrees. When a captive pedigree is inaccurate or incomplete, molecular data have the potential to improve breeding program management. Molecular markers can

Exploration of a rare population of Chinese chestnut in North America: Stand dynamics, health, and genetic relationships

With worldwide transport of plants, exotic species spread disease to native relatives, but they can also provide disease resistance via hybrid breeding programs. American chestnut was nearly eliminated from North America by introduced chestnut blight, but resistant hybrids of American and Chinese chestnuts are being created. We studied the ecology and genetics of an unmanaged population of Chinese chestnut in North America. The forest dynamics of pure Chinese chestnut indicate that hybrids between the Chinese and American species could be successful colonizers of forests in North America. We challenge the paradigm that exotic tree species are wholly detrimental to native biodiversity.

Variation in needle and cone traits in natural populations of Pinus yunnanensis

Pinus yunnanensis Franch. is a major component of coniferous forests in southwestern China. Little is known about its intraspecific variation. Morphological variations in needle and cone traits of P. yunnanensis were analyzed to quantify variability among and within natural populations. Seven traits were measured on 10 needles collected from 30 trees in each of the 18 sampled populations of P. yunnanensis. Four cone traits were measured in 221 individual trees from nine populations. The results showed that there were significant differences (pxa0<xa00.01) both among populations and within populations in each needle and cone trait. The proportion of phenotypic variation of nearly all needle and cone traits was over 50xa0% within populations, which showed trees within populations accounted for a majority of the total variation. The needle traits showed higher variability within population than cone traits. Variability in the needle traits was correlated with geo-climatic parameters (longitude, latitude, altitude, temperature, and precipitation). Needle length and the ratio of needle length to fascicle sheath length showed clinal variation in response to latitudinal and altitudinal gradients. A hierarchical classification of all populations based on needle traits led to the formation of four major groups. The findings provide important genetic information for the evaluation of variation. Moreover, it will assist in management of genetic diversity of P. yunnanensis.

Population structure and genotypic variation of Crataegus pontica inferred by molecular markers.

Information about the natural patterns of genetic variability and their evolutionary bases are of fundamental practical importance for sustainable forest management and conservation. In the present study, the genetic diversity of 164 individuals from fourteen natural populations of Crataegus pontica K.Koch was assessed for the first time using three genome-based molecular techniques; inter-retrotransposon amplified polymorphism (IRAP); inter-simple sequence repeats (ISSR) and start codon targeted (SCoT) polymorphism. IRAP, ISSR and SCoT analyses yielded 126, 254 and 199 scorable amplified bands, respectively, of which 90.48, 93.37 and 83.78% were polymorphic. ISSR revealed efficiency over IRAP and SCoT due to high effective multiplex ratio, marker index and resolving power. The dendrograms based on the markers used and combined data divided individuals into three major clusters. The correlation between the coefficient matrices for the IRAP, ISSR and SCoT data was significant. A higher level of genetic variation was observed within populations than among populations based on the markers used. The lower divergence levels depicted among the studied populations could be seen as evidence of gene flow. The promotion of gene exchange will be very beneficial to conserve and utilize the enormous genetic variability.

Population genetics, phylogenomics and hybrid speciation of Juglans in China determined from whole chloroplast genomes, transcriptomes, and genotyping-by-sequencing (GBS)

Genomic data are a powerful tool for elucidating the processes involved in the evolution and divergence of species. The speciation and phylogenetic relationships among Chinese Juglans remain unclear. Here, we used results from phylogenomic and population genetic analyses, transcriptomics, Genotyping-By-Sequencing (GBS), and whole chloroplast genomes (Cp genome) data to infer processes of lineage formation among the five native Chinese species of the walnut genus (Juglans, Juglandaceae), a widespread, economically important group. We found that the processes of isolation generated diversity during glaciations, but that the recent range expansion of J. regia, probably from multiple refugia, led to hybrid formation both within and between sections of the genus. In southern China, human dispersal of J. regia brought it into contact with J. sigillata, which we determined to be an ecotype of J. regia that is now maintained as a landrace. In northern China, walnut hybridized with a distinct lineage of J. mandshurica to form J. hopeiensis, a controversial taxon (considered threatened) that our data indicate is a horticultural variety. Comparisons among whole chloroplast genomes and nuclear transcriptome analyses provided conflicting evidence for the timing of the divergence of Chinese Juglans taxa. J. cathayensis and J. mandshurica are poorly differentiated based our genomic data. Reconstruction of Juglans evolutionary history indicate that episodes of climatic variation over the past 4.5 to 33.80 million years, associated with glacial advances and retreats and population isolation, have shaped Chinese walnut demography and evolution, even in the presence of gene flow and introgression.

De novo genome assembly of Geosmithia morbida, the causal agent of thousand cankers disease.

Geosmithia morbida is a filamentous ascomycete that causes thousand cankers disease in the eastern black walnut tree. This pathogen is commonly found in the western U.S.; however, recently the disease was also detected in several eastern states where the black walnut lumber industry is concentrated. G. morbida is one of two known phytopathogens within the genus Geosmithia, and it is vectored into the host tree via the walnut twig beetle. We present the first de novo draft genome of G. morbida. It is 26.5 Mbp in length and contains less than 1% repetitive elements. The genome possesses an estimated 6,273 genes, 277 of which are predicted to encode proteins with unknown functions. Approximately 31.5% of the proteins in G. morbida are homologous to proteins involved in pathogenicity, and 5.6% of the proteins contain signal peptides that indicate these proteins are secreted. Several studies have investigated the evolution of pathogenicity in pathogens of agricultural crops; forest fungal pathogens are often neglected because research efforts are focused on food crops. G. morbida is one of the few tree phytopathogens to be sequenced, assembled and annotated. The first draft genome of G. morbida serves as a valuable tool for comprehending the underlying molecular and evolutionary mechanisms behind pathogenesis within the Geosmithia genus.

Estimating heritability of disease resistance and factors that contribute to long-term survival in butternut (Juglans cinerea L.)

For most wild species affected by exotic pests or pathogens, the relative importance of heritable genetic differences in determining apparent variation in disease resistance is unknown. This is true in particular for butternut, a North American hardwood affected by butternut canker disease and undergoing demographic contraction. Little is known about site effects on butternut decline, in part because long-term monitoring data are lacking. We collected detailed disease phenotypes and multilocus microsatellite genotypes for all surviving individuals in a large natural population of butternut in 2003 (nu2009=u2009302) and 2012 (nu2009=u2009113). Two analytical methods, correlations between pairwise phenotypic similarity and pairwise relatedness, and estimation of among-family variance, both indicated weak heritability of disease-related traits and no heritability for overall tree health in the population. Additionally, an analysis of spatial data collected in 2001 (nu2009=u2009341) and 2012 (nu2009=u2009113) demonstrated that drier, upland sites contribute to increased likelihood of survival. We conclude that genetic differences among wild butternut individuals contributed little to observed variance in survival over 10xa0years but fine-scale site differences were useful predictors of butternut mortality.

Localized gene expression changes during adventitious root formation in black walnut (Juglans nigra L.)

Cutting propagation plays a large role in the forestry and horticulture industries where superior genotypes need to be clonally multiplied. Integral to this process is the ability of cuttings to form adventitious roots. Recalcitrance to adventitious root development is a serious hurdle for many woody plant propagation systems including black walnut (Juglans nigra L.), an economically valuable species. The inability of black walnut to reliably form adventitious roots limits propagation of superior genotypes. Adventitious roots originate from different locations, and root induction is controlled by many environmental and endogenous factors. At the molecular level, however, the regulation of adventitious root formation is still poorly understood. In order to elucidate the transcriptional changes during adventitious root development in black walnut, we used quantitative real-time polymerase chain reaction to measure the expression of nine key genes regulating root formation in other species. Using our previously developed spatially explicit timeline of adventitious root development in black walnut softwood cuttings, we optimized a laser capture microdissection protocol to isolate RNA from cortical, phloem fiber and phloem parenchyma cells throughout adventitious root formation. Laser capture microdissection permitted high-resolution, site-specific analysis of gene expression that differentiated between participatory and non-participatory root progenitor cells. Results indicated mRNA abundance was altered in all nine rooting-related genes in response to auxin treatment in both juvenile and mature cuttings. SCARECROW LIKE-1 (SCL) had the greatest change in expression in juvenile rooting-competent cells at days 16 and 18, with a 24- and 23-fold increase relative to day 0, respectively. Tissues not linked to root organogenesis had little change in SCL expression at similar time points. AUXIN RESPONSE FACTOR (ARF)6 and ARF8 as well as SHORTROOT expression also increased 2- to 4-fold in rooting-competent tissue. The greatest transcript abundance in rooting-competent cuttings was restricted to root progenitor cells, while recalcitrant cuttings had a diffuse mRNA signal among tissue types.