Biocultural diversity of common walnut (Juglans regia L.) and sweet chestnut (Castanea sativa Mill.) across Eurasia.

A biocultural diversity approach integrates plant biology and germplasm dispersal processes with human cultural diversity. An increasing number of studies have identified cultural factors and ethnolinguistic barriers as the main drivers of the genetic diversity in crop plants. Little is known about how anthropogenic processes have affected the evolution of tree crops over the entire time scale of their interaction with humans. In Asia and the Mediterranean, common walnut (Juglans regia L.) and sweet chestnut (Castanea sativa Mill.) have been economically and culturally important crops for millennia; there, in ancient times, they were invested with symbolic and religious significance. In this study, we detected a partial geographic congruence between the ethno-linguistic repartition of human communities, the distribution of major cognitive sets of word-related terms, and the inferred genetic clusters of common walnut and sweet chestnut populations across Eurasia. Our data indicated that isolation by distance processes, landscape heterogeneity and cultural boundaries might have promoted simultaneously human language diversification and walnut/chestnut differentiation across the same geographic macro-regions. Hotspots of common walnut and sweet chestnut genetic diversity were associated with areas of linguistic enrichment in the Himalayas, Trans-Caucasus, and Pyrenees Mountains, where common walnuts and sweet chestnuts had sustained ties to human culture since the Early Bronze Age. Our multidisciplinary approach supported the indirect and direct role of humans in shaping walnut and chestnut diversity across Eurasia from the EBA (e.g., Persian Empire and Greek-Roman colonization) until the first evidence of active selection and clonal propagation by grafting of both species. Our findings highlighted the benefit of an efficient integration of the relevant cultural factors in the classical genome (G) × environmental (E) model and the urgency of a systematic application of the biocultural diversity concept in the reconstruction of the evolutionary history of tree species.

Phylogenomics Reveals an Ancient Hybrid Origin of the Persian Walnut.

Persian walnut (Juglans regia) is cultivated worldwide for its high-quality wood and nuts, but its origin has remained mysterious because in phylogenies it occupies an unresolved position between American black walnuts and Asian butternuts. Equally unclear is the origin of the only American butternut, J. cinerea. We resequenced the whole genome of 80 individuals from 19 of the 22 species of Juglans and assembled the genome of its relatives Pterocarya stenoptera and Platycarya strobilacea. Using phylogenetic-network analysis of single-copy nuclear genes, genome-wide site pattern probabilities, and Approximate Bayesian Computation, we discovered that J. regia (and its landrace J. sigillata) arose as a hybrid between the American and the Asian lineages and that J. cinerea resulted from massive introgression from an immigrating Asian butternut into the genome of an American black walnut. Approximate Bayesian Computation modeling placed the hybrid origin in the late Pliocene, ∼3.45 My, with both parental lineages since having gone extinct in Europe.

Microsatellite Borders and Micro-sequence Conservation in Juglans.

Walnuts (Juglans spp.) are economically important nut and timber species with a worldwide distribution. Using the published Persian walnut genome as a reference for the assembly of short reads from six Juglans species and several interspecific hybrids, we identified simple sequence repeats in 12 Juglans nuclear and organellar genomes. The genome-wide distribution and polymorphisms of nuclear and organellar microsatellites (SSRs) for most Juglans genomes have not been previously studied. We compared the frequency of nuclear SSR motifs and their lengths across Juglans, and identified section-specific chloroplast SSR motifs. Primer pairs were designed for more than 60,000 SSR-containing sequences based on alignment against assembled scaffold sequences. Of the >60,000 loci, 39,000 were validated by e-PCR using unique primer pairs. We identified primers containing 100% sequence identity in multiple species. Across species, sequence identity in the SSR-flanking regions was generally low. Although SSRs are common and highly dispersed in the genome, their flanking sequences are conserved at about 90 to 95% identity within Juglans and within species. In a few rare cases, flanking sequences are identical across species of Juglans. This comprehensive report of nuclear and organellar SSRs in Juglans and the generation of validated SSR primers will be a useful resource for future genetic analyses, walnut breeding programs, high-level taxonomic evaluations, and genomic studies in Juglandaceae.

Pooled whole-genome sequencing of interspecific chestnut (Castanea) hybrids reveals loci associated with differences in caching behavior of fox squirrels (Sciurus niger L.).

Dispersal of seeds by scatter-hoarding rodents is common among tropical and temperate tree species, including chestnuts in the genus Castanea. Backcrossed (BC) interspecific hybrid chestnuts exhibit wide variation in seed traits: as the parent species (Castanea dentata and C. mollissima) have distinct seed phenotypes and tend to be handled differently by seed dispersers, phenotypic variation in BC trees is likely due to inheritance of genes that have undergone divergent evolution in the parent species. To identify candidate genomic regions for interspecific differences in seed dispersal, we used tagged seeds to measure average dispersal distance for seeds of third-generation BC chestnuts and sequenced pooled whole genomes of mother trees with contrasting seed dispersal: high caching rate/long distance; low caching rate/short distance; no caching. Candidate regions affecting seed dispersal were identified as loci with more C. mollissima alleles in the high caching rate/ long-distance pool than expected by chance and observed in the other two pools. Functional annotations of candidate regions included predicted lipid metabolism, dormancy regulation, seed development, and carbohydrate metabolism genes. The results support the hypothesis that perception of seed dormancy is a predominant factor in squirrel caching decisions, and also indicate profitable directions for future work on the evolutionary genomics of trees and coevolved seed dispersers.

Pollen flow and paternity in an isolated and non-isolated black walnut (Juglans nigra L.) timber seed orchard.

Artificial pollination of black walnut (Juglans nigra L.) is not practical and timber breeders have historically utilized only open-pollinated half-sib families. An alternate approach called "breeding without breeding," consists of genotyping open-pollinated progeny using DNA markers to identify paternal parents and then constructing full-sib families. In 2014, we used 12 SSR markers to genotype 884 open-pollinated half-sib progeny harvested from two clonal orchards containing 206 trees, comprised of 52 elite timber selections. Seed was harvested in 2011 from each of two ramets of 23 clones, one upwind and one downwind, based on prevailing wind direction from the west-southwest. One orchard was isolated from wild black walnut and composed of forward selections while the other orchard was adjacent to a natural forest containing mature black walnut composed of backward selections. Isolation significantly increased within-orchard pollination (85%) of the progeny from the isolated orchard compared to 42% from the non-isolated orchard. Neither prevailing wind direction nor seed tree position in the orchard affected paternity patterns or wild pollen contamination. Genetic diversity indices revealed that progeny from both orchards were in Hardy-Weinberg equilibrium with very little inbreeding and no selfing. A significant level of inbreeding was present among the forward selected parents, but not the first generation (backward selected) parents. Some orchard clones failed to sire any progeny while other clones pollinated upwards of 20% of progeny.

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.

Demographically idiosyncratic responses to climate change and rapid Pleistocene diversification of the walnut genus Juglans (Juglandaceae) revealed by whole-genome sequences.

Whether species demography and diversification are driven primarily by extrinsic environmental changes such as climatic oscillations in the Quaternary or by intrinsic biological interactions like coevolution between antagonists is a matter of active debate. In fact, their relative importance can be assessed by tracking past population fluctuations over considerable time periods. We applied the pairwise sequentially Markovian coalescent approach on the genomes of 11 temperate Juglans species to estimate trajectories of changes in effective population size (Ne ) and used a Bayesian-coalescent based approach that simultaneously considers multiple genomes (G-PhoCS) to estimate divergence times between lineages. Ne curves of all study species converged 1.0 million yr ago, probably reflecting the time when the walnut genus last shared a common ancestor. This estimate was confirmed by the G-PhoCS estimates of divergence times. But all species did not react similarly to the dramatic climatic oscillations following early Pleistocene cooling, so the timing and amplitude of changes in Ne differed among species and even among conspecific lineages. The population histories of temperate walnut species were not driven by extrinsic environmental changes alone, and a key role was probably played by species-specific factors such as coevolutionary interactions with specialized pathogens.

Vegetative and Adaptive Traits Predict Different Outcomes for Restoration Using Hybrids.

Hybridization has been implicated as a driver of speciation, extinction, and invasiveness, but can also provide resistant breeding stock following epidemics. However, evaluating the appropriateness of hybrids for use in restoration programs is difficult. Past the F1 generation, the proportion of a progenitor's genome can vary widely, as can the combinations of parental genomes. Detailed genetic analysis can reveal this information, but cannot expose phenotypic alterations due to heterosis, transgressive traits, or changes in metabolism or development. In addition, because evolution is often driven by extreme individuals, decisions based on phenotypic averages of hybrid classes may have unintended results. We demonstrate a strategy to evaluate hybrids for use in restoration by visualizing hybrid phenotypes across selected groups of traits relative to both progenitor species. Specifically, we used discriminant analysis to differentiate among butternut (Juglans cinerea L.), black walnut (J. nigra L.), and Japanese walnut (J. ailantifolia Carr. var. cordiformis) using vegetative characters and then with functional adaptive traits associated with seedling performance. When projected onto the progenitor trait space, naturally occurring hybrids (J. × bixbyi Rehd.) between butternut and Japanese walnut showed introgression toward Japanese walnut at vegetative characters but exhibited a hybrid swarm at functional traits. Both results indicate that hybrids have morphological and ecological phenotypes that distinguish them from butternut, demonstrating a lack of ecological equivalency that should not be carried into restoration breeding efforts. Despite these discrepancies, some hybrids were projected into the space occupied by butternut seedlings' 95% confidence ellipse, signifying that some hybrids were similar at the measured traits. Determining how to consistently identify these individuals is imperative for future breeding and species restoration efforts involving hybrids. Discriminant analysis provides a useful technique to visualize past selection mechanisms and current variation in hybrid populations, especially when key ecological traits that distinguish progenitors are unknown. Furthermore, discriminant analysis affords a tool to assess ecological equivalency of hybrid populations and breeding program efforts to select for certain traits and monitor the amount of variability of those traits, relative to progenitors.

De novo assembly and characterization of the leaf, bud, and fruit transcriptome from the vulnerable tree Juglans mandshurica for the development of 20 new microsatellite markers using Illumina sequencing.

Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.

The complete chloroplast genome of common walnut (Juglans regia).

Common walnut (Juglans regia L.) is cultivated in temperate regions worldwide for its wood and nuts. The complete chloroplast genome of J. regia was sequenced using the Illumina MiSeq platform. This is the first complete chloroplast sequence for the Juglandaceae, a family that includes numerous species of economic importance. The chloroplast genome of J. regia was 160 367 bp in length, with 36.11% GC content. It contains a pair of inverted repeats (26 035 bp) which were separated by a large single copy (89 872 bp) and a small single copy region (18 425 bp). A total of 137 genes were annotated, which included 86 protein-coding genes, three pseudogenes (two ycf15 and one infA), 40 tRNA genes and eight rRNA genes. The neighbour-joining phylogenetic analysis with the reported chloroplast genomes showed that common walnut chloroplasts are most closely related to those of the Fagaceae family.

Completion of the Chloroplast Genomes of Five Chinese Juglans and Their Contribution to Chloroplast Phylogeny.

Juglans L. (walnuts and butternuts) is an economically and ecologically important genus in the family Juglandaceae. All Juglans are important nut and timber trees. Juglans regia (Common walnut), J. sigillata (Iron walnut), J. cathayensis (Chinese walnut), J. hopeiensis (Ma walnut), and J. mandshurica (Manchurian walnut) are native to or naturalized in China. A strongly supported phylogeny of these five species is not available due to a lack of informative molecular markers. We compared complete chloroplast genomes and determined the phylogenetic relationships among the five Chinese Juglans using IIumina sequencing. The plastid genomes ranged from 159,714 to 160,367 bp encoding 128 functional genes, including 88 protein-coding genes and 40 tRNA genes each. A complete map of the variability across the genomes of the five Juglans species was produced that included single nucleotide variants, indels (insertions and deletions), and large structural variants, as well as differences in simple sequence repeats (SSR) and repeat sequences. Molecular phylogeny strongly supported division of the five walnut species into two previously recognized sections (Juglans/Dioscaryon and Cardiocaryon) with a 100% bootstrap (BS) value using the complete cp genomes, protein coding sequences (CDS), and the introns and spacers (IGS) data. The availability of these genomes will provide genetic information for identifying species and hybrids, taxonomy, phylogeny, and evolution in Juglans, and also provide insight into utilization of Juglans plants.

Ancient Humans Influenced the Current Spatial Genetic Structure of Common Walnut Populations in Asia.

Common walnut (Juglans regia L) is an economically important species cultivated worldwide for its wood and nuts. It is generally accepted that J. regia survived and grew spontaneously in almost completely isolated stands in its Asian native range after the Last Glacial Maximum. Despite its natural geographic isolation, J. regia evolved over many centuries under the influence of human management and exploitation. We evaluated the hypothesis that the current distribution of natural genetic resources of common walnut in Asia is, at least in part, the product of ancient anthropogenic dispersal, human cultural interactions, and afforestation. Genetic analysis combined with ethno-linguistic and historical data indicated that ancient trade routes such as the Persian Royal Road and Silk Road enabled long-distance dispersal of J. regia from Iran and Trans-Caucasus to Central Asia, and from Western to Eastern China. Ancient commerce also disrupted the local spatial genetic structure of autochthonous walnut populations between Tashkent and Samarkand (Central-Eastern Uzbekistan), where the northern and central routes of the Northern Silk Road converged. A significant association between ancient language phyla and the genetic structure of walnut populations is reported even after adjustment for geographic distances that could have affected both walnut gene flow and human commerce over the centuries. Beyond the economic importance of common walnut, our study delineates an alternative approach for understanding how the genetic resources of long-lived perennial tree species may be affected by the interaction of geography and human history.

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.

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

With the transport of plants around the globe, exotic species can readily spread disease to their native relatives; however, they can also provide genetic resistance to those relatives through hybrid breeding programmes. American chestnut (Castanea dentata) was an abundant tree species in North America until its decimation by introduced chestnut blight. To restore chestnut in North America, efforts are ongoing to test putative blight-resistant hybrids of Castanea dentata and Chinese chestnut (Castanea mollissima), but little is known about the ecology of C. mollissima. In a forest in northeastern USA in which C. mollissima has become established, we explored questions of stand dynamics, health and genetic relationships of C. mollissima offspring to an adjacent parent orchard. We found that C. mollissima was adapted and randomly distributed among native species in this relatively young forest. The genetics of the C. mollissima population compared with its parents indicated little effect of selection pressure as each of the parent trees contributed at least one offspring. The ease with which this exotic species proliferated calls to question why C. mollissima is rare elsewhere in forests of North America. It is likely that a time window of low animal predation allowed seedlings to establish, and the shallow soil at this site limited the maximum forest canopy height, permitting the characteristically short-statured C. mollissima to avoid suppression. Our results indicate that because C. mollissima exhibited pioneer species characteristics, hybrids between C. mollissima and C. dentata have the potential to be successful pioneer species of future forests in North America, and we challenge the paradigm that exotic tree species are wholly detrimental to native biodiversity. We contend that exotic tree species should be assessed not only by their level of threat to native species, but also by their potential positive impacts on ecosystems via hybrid breeding programmes.

Improving disease resistance of butternut (Juglans cinerea), a threatened fine hardwood: a case for single-tree selection through genetic improvement and deployment.

Approaches for the development of disease-resistant butternut (Juglans cinerea L.) are reviewed. Butternut is a threatened fine hardwood throughout its natural range in eastern North America because of the invasion of the exotic fungus, Sirococcus clavigignenti-juglandacearum Nair, Kostichka and Kuntz, which causes butternut canker. Early efforts were made to identify and collect putatively resistant germ plasm, identify vectors and to characterize the disease. More recently, molecular techniques have been employed to genetically characterize both the pathogen and the resistant germ plasm. Much of the host resistance may originate from hybridization with a close Asian relative, Japanese walnut (Juglans ailanthifolia Carr.), and from a few natural phenotypic variants. Further genetic characterization is needed before classical breeding or genetic modification can be used to produce canker-resistant trees.