Morpho-Physiological and Genomic Evaluation of Juglans Species Reveals Regional Maladaptation to Cold Stress.

Climate change may have unpredictable effects on the cold hardiness of woody species planted outside of their range of origin. Extreme undulations in temperatures may exacerbate susceptibility to cold stress, thereby interfering with productivity and ecosystem functioning. Juglans L. and their naturally occurring interspecific F1 hybrids, are distributed natively across many temperate regions, and J. regia has been extensively introduced. Cold hardiness, an environmental and genetic factor yet to be evaluated in many native and introduced Juglans species, may be a limiting factor under future climate change and following species introductions. We evaluated cold hardiness of native North American and Eastern Asian Juglans along with J. regia genotypes using field data from the Midwestern United States (Indiana), controlled freezing tests, and genome sequencing with close assessment of Juglans cold hardy genes. Many Juglans species previously screened for cold-hardiness were genotypes derived from the Midwest, California, and Europe. In 2014, despite general climate adaptation, Midwestern winter temperatures of -30°C killed J. regia originating from California; however, naturalized Midwestern J. regia survived and displayed low damage. Hybridization of J. regia with black walnut (J. nigra) and butternut (J. cinerea) produced F1s displaying greater cold tolerance than pure J. regia. Cold hardiness and growth are variable in Midwestern J. regia compared to native Juglans, East Asian Juglans, and F1 hybrids. Phylogeny analyses revealed that J. cinerea sorted with East Asian species using the nuclear genome but with North American species using the organellar genome. Investigation of selected cold hardy genes revealed that J. regia was distinct from other species and exhibited less genetic diversity than native Juglans species Average whole genome heterozygosity and Tajima's D for cold hardy genes was low within J. regia samples and significantly higher for hybrid as well as J. nigra. We confirmed that molecular and morpho-physiological data were highly correlated and thus can be used effectively to characterize cold hardiness in Juglans species. We conclude that the genetic diversity within local J. regia populations is low and additional germplasm is needed for development of more regionally adapted J. regia varieties.

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.

The miR164-dependent regulatory pathway in developing maize seed.

MicroRNA164 (miR164) plays a key role in leaf and flower development, lateral root initiation, and stress responses. However, little is known about the regulatory roles of miR164 during seed development, particularly in maize. The aim of this study was to discover the developmental function of miR164 in maize seed. Small RNA sequencing (sRNA-seq) was performed at two key stages. The results indicated that miR164 was down-regulated during maize seed development. In addition, degradome library sequencing and transient expression assays identified the target genes for miR164. Two microRNA (miRNA) pairs, miR164-NAM, ATAF, and CUC32 (NAC32) and miR164-NAC40, were isolated. The developmental function of miR164 was determined by analyzing the differentially expressed genes (DEGs) between the wild-type and miR164 transgenic lines using RNA sequencing (RNA-seq) and by screening the DEGs related to NAC32 and NAC40 via co-expression and transient expression analysis. These results identified two beta-expansin genes, EXPB14 and EXPB15, which were located downstream of the NAC32 and NAC40 genes. This study revealed, for the first time, a miR164-dependent regulatory pathway, miR164-NAC32/NAC40-EXPB14/EXPB15, which participates in maize seed expansion. These findings highlight the significance of miR164 in maize seed development, and can be used to explore the role of miRNA in seed development.

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.

Overexpression of AtSTO1 leads to improved salt tolerance in Populus tremula × P. alba.

One of the major abiotic stress conditions limiting healthy growth of trees is salinity stress. The use of gene manipulation for increased tolerance to abiotic stress has been successful in many plant species. Overexpression of the Arabidopsis SALT TOLERANT1 (STO1) gene leads to increased concentrations of 9-cis-epoxycarotenoid dioxygenase3, a vital enzyme in Arabidopsis abscisic acid biosynthesis. In the present work, the Arabidopsis STO1 gene (AtSTO1) was overexpressed in poplar to determine if the transgene would confer enhanced salt tolerance to the generated transgenics. The results of multiple greenhouse trials indicated that the transgenic poplar lines had greater levels of resistance to NaCl than wild-type plants. Analysis using RT-PCR indicated a variation in the relative abundance of the STO1 transcript in the transgenics that coincided with tolerance to salt. Several physiological and morphological changes such as greater overall biomass, greater root biomass, improved photosynthesis, and greater pith size were observed in the transgenics when compared to controls undergoing salt stress. These results indicated overexpression of AtSTO1 improved salt tolerance in poplar.

Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response.

Primary cilia and basal bodies are evolutionarily conserved organelles that mediate communication between the intracellular and extracellular environments. Here we show that bbs1, bbs4 and mkks (also known as bbs6), which encode basal body proteins, are required for convergence and extension in zebrafish and interact with wnt11 and wnt5b. Suppression of bbs1, bbs4 and mkks transcripts results in stabilization of beta-catenin with concomitant upregulation of T-cell factor (TCF)-dependent transcription in both zebrafish embryos and mammalian ciliated cells, a defect phenocopied by the silencing of the axonemal kinesin subunit KIF3A but not by chemical disruption of the cytoplasmic microtubule network. These observations are attributable partly to defective degradation by the proteasome; suppression of BBS4 leads to perturbed proteasomal targeting and concomitant accumulation of cytoplasmic beta-catenin. Cumulatively, our data indicate that the basal body is an important regulator of Wnt signal interpretation through selective proteolysis and suggest that defects in this system may contribute to phenotypes pathognomonic of human ciliopathies.