Chemical profile and analysis of biosynthetic pathways and genes of volatile terpenes in Pityopsis ruthii, a rare and endangered flowering plant.

It is critical to gather biological information about rare and endangered plants to incorporate into conservation efforts. The secondary metabolism of Pityopsis ruthii, an endangered flowering plant that only occurs along limited sections of two rivers (Ocoee and Hiwassee) in Tennessee, USA was studied. Our long-term goal is to understand the mechanisms behind P. ruthii's adaptation to restricted areas in Tennessee. Here, we profiled the secondary metabolites, specifically in flowers, with a focus on terpenes, aiming to uncover the genomic and molecular basis of terpene biosynthesis in P. ruthii flowers using transcriptomic and biochemical approaches. By comparative profiling of the nonpolar portion of metabolites from various tissues, P. ruthii flowers were rich in terpenes, which included 4 monoterpenes and 10 sesquiterpenes. These terpenes were emitted from flowers as volatiles with monoterpenes and sesquiterpenes accounting for almost 68% and 32% of total emission of terpenes, respectively. These findings suggested that floral terpenes play important roles for the biology and adaptation of P. ruthii to its limited range. To investigate the biosynthesis of floral terpenes, transcriptome data for flowers were produced and analyzed. Genes involved in the terpene biosynthetic pathway were identified and their relative expressions determined. Using this approach, 67 putative terpene synthase (TPS) contigs were detected. TPSs in general are critical for terpene biosynthesis. Seven full-length TPS genes encoding putative monoterpene and sesquiterpene synthases were cloned and functionally characterized. Three catalyzed the biosynthesis of sesquiterpenes and four catalyzed the biosynthesis of monoterpenes. In conclusion, P. ruthii plants employ multiple TPS genes for the biosynthesis of a mixture of floral monoterpenes and sesquiterpenes, which probably play roles in chemical defense and attracting insect pollinators alike.

Development and Characterization of 20 Genomic SSR Markers for Ornamental Cultivars of Weigela.

Weigela (Caprifoliaceae) is a genus of ornamental plants popular for its phenotypic variation and hardiness, that includes species hybridized to produce the commercially available cultivars. Despite its popularity, limited genetic resources exist for the genus. Twenty genomic simple sequence repeat (gSSR) markers distributed across the genome were developed using low coverage whole-genome sequencing data of Weigela Spilled Wine®. A cross-amplification evaluation with these 20 gSSR markers on a collection of 18 Weigela cultivars revealed a total of 111 unique alleles, including 36 private alleles. A diagrammatic key was constructed to identify cultivars using only six of the gSSR markers, demonstrating the newly developed gSSR markers are immediately useful for cultivar identification. Future uses could include breeding with marker-assisted selection, determining the history of hybridization of the current cultivated lines, aiding in the construction of genetic maps, and assessing the patterns of population genetic structure of Weigela spp.

Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Chloroplast genome of the invasive Pyrus calleryana underscores the high molecular diversity of the species.

The complete chloroplast genome of Pyrus calleryana (GenBank OM541581.1) was developed by de novo assembly from whole-genome sequencing data. Reference-guided (P. phaeocarpa) read mapping and assembly were followed by annotation and phylogenetic comparisons. The 159,965 bp P. calleryana chloroplast genome represented 36.56% GC content with a classical quadripartite architecture and two inverted repeats regions (IRs; each 26,392 bp) separating the large single-copy region (LSC; 87,942 bp) and the small single-copy region (SSC; 19.239 bp). In total, 125 unique features were annotated in that genome, including 83 protein coding genes, 38 tRNA coding genes, and 4 rRNA coding genes. Phylogenetic analyses based on the whole chloroplast genome sequences placed the P. calleryana among other Rosaceae plants, specifically among the Asian species of Pyrus.

Development and Characterization of 15 Novel Genomic SSRs for Viburnum farreri.

The Viburnum genus is of particular interest to horticulturalists, phylogeneticists, and biogeographers. Despite its popularity, there are few existing molecular markers to investigate genetic diversity in this large genus, which includes over 160 species. There are also few polymorphic molecular tools that can delineate closely related species within the genus. Viburnum farreri, a member of the Solenotinus subclade and one of the centers of diversity for Viburnum, was selected for DNA sequencing and development of genomic simple sequence repeats (gSSRs). In this study, 15 polymorphic gSSRs were developed and characterized for a collection of 19 V. farreri samples. Number of alleles per locus ranged from two- to- eight and nine loci had four or more alleles. Observed heterozygosity ranged from 0 to 0.84 and expected heterozygosity ranged from 0.10 to 0.80 for the 15 loci. Shannon diversity index values across these loci ranged from 0.21 to 1.62. The markers developed in this study add to the existing molecular toolkit for the genus and will be used in future studies investigating cross-transferability, genetic variation, and species and cultivar delimitation in the Viburnum genus and closely allied genera in the Adoxaceae and Caprifoliaceae.

Haplotyping of Cornus florida and C. kousa chloroplasts: Insights into species-level differences and patterns of plastic DNA variation in cultivars.

Chloroplast DNA is a part of plant non-nuclear genome, and is of particular interest for lineage studies. Moreover, the non-coding regions of cpDNA display higher mutation rates than the conserved coding cpDNA, which has been employed for phylogenetic and population research. We analyzed the cpDNA of 332 gDNA samples from collections of Cornus florida and C. kousa (commercial cultivars, breeding selections, and wild kousa accessions from Asia), using the chlorotyping system developed on North America-native, wild accessions of C. florida. Our results indicated significant differences in chlorotype frequencies between the two species. Cornus florida samples were represented by all major chlorotypes previously described, whereas all C. kousa samples analyzed had only one of the chlorotype patterns shown by C. florida. The chlorotyping analytic panel was then expanded by sequencing the targeted three non-coding cpDNA regions. Results indicated a major difference in the maternally-inherited cpDNA between the two closely related Big-Bracted Cornus species. Chlorotype diversity and differences in the proportion of informative sites in the cpDNA regions of focus emphasized the importance of proper loci choice for cpDNA-based comparative studies between the closely related dogwood species. Phylogenetic analyses of the retrieved sequences for the other species of Cornus provided information on the relative utility of the cpDNA regions studied and helped delineate the groups (Big-Bracted, Cornelian Cherries, Blue/White-Fruited) within the genus. Genealogical relationships based on the cpDNA sequences and the inferred chlorotype networks indicated the need for continued analyses across further non-coding cpDNA regions to improve the phylogenetic resolution of dogwoods.