High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.

Stable genetic resistance to the fungal disease eastern filbert blight (EFB), caused by Anisogramma anomala, is vital for sustainable production of European hazelnut (Corylus avellana) in eastern North America. In this study, new hazelnut germplasm from the Russian Federation, Ukraine, and Poland (a total of 1844 trees from 66 seed lots) was subjected to A. anomala under field conditions over at least five years in New Jersey. Plants were then rated for the presence of EFB using an index of 0 (no disease) through 5 (all stems containing cankers). Nuts of the resistant trees were evaluated to identify plants with improved kernel characteristics. Genomic DNA of these trees was also screened with sequence-characterized amplified region (SCAR) markers generated by the primers BE-03, BE-33, and BE-68, which are closely linked to the single dominant R-gene of ‘Gasaway’, to assess the resistant seedlings for the presence of this well-known source of resistance. At final evaluation, 76 trees remained free of disease with nine expressing only minor symptoms (rating 1 or 2). The resistant trees spanned 24 different seed lots representing all three countries. The remaining trees ranged from moderately to severely infected with 81% of the total collection rating 5. Several of the resistant trees were found to produce commercial-sized (≈12 mm diameter), round kernels that blanched well. Although the results of the ‘Gasaway’ SCAR primers were inconclusive, the diverse collection origins and disease phenotypes provide evidence that novel sources of resistance were likely identified in this study. These new plants should broaden the genetic base of EFB-resistant C. avellana hazelnut germplasm available for breeding.

Endophytic bacteria have been shown to provide several advantages to their host, including enhanced growth. Inoculating biofuel species with endophytic bacteria is therefore an attractive option to increase the productivity of biofuel feedstocks. Here, we investigated the effect of inoculating hard wood cuttings of Populus deltoides Bartr. 9 Populus. nigra L. clone OP367 with Enterobacter sp. 638. After 17 weeks, plants inoculated with Enterobacter sp. 638 had 55% greater total biomass than un-inoculated control plants. Study of gas exchange and fluorescence in developing and mature leaves over a diurnal cycle and over a 5 week measurement campaign revealed no effects of inoculation on photosynthesis, stomatal conductance, photosynthetic water use efficiency or the maximum and operating efficiency of photosystem II. However, plants inoculated with Enterobacter sp. 638 had a canopy that was 39% larger than control plants indicating that the enhanced growth was fueled by increased leaf area, not by improved physiology. Leaf nitrogen content was determined at two stages over the 5 week measurement period. No effect of Enterobacter sp. 638 on leaf nitrogen content was found indicating that the larger plants were acquiring sufficient nitrogen. Enterobacter sp. 638 lacks the genes for N 2 fixation, therefore the increased availability of nitrogen likely resulted from enhanced nitrogen acquisition by the 84% larger root system. These data show that Enterobacter sp. 638 has the potential to dramatically increase productivity in poplar. If fully realized in the production environment, these results indicate that an increase in the environmental and economic viability of poplar as a biofuel feedstock is possible when inoculated with endophytic bacteria like Enterobacter sp. 638.

The development of new cultivars resistant to the disease eastern filbert blight (EFB), caused by Anisogramma anomala, is of primary importance to hazelnut (Corylus sp.) breeders in North America. Recently, a large number of EFB-resistant cultivars, grower selections, and seedlings from foreign germplasm collections were identified. However, for a significant number of these, little is known of their origin, relationships, or genetic background. In this study, 17 microsatellite markers were used to investigate the genetic diversity and population structure of 323 unique accessions, including EFB-resistant and tolerant germplasm of uncertain origins, in comparison with a panel of known reference accessions representing a wide diversity of Corylus cultivars, breeding selections, and interspecific hybrids. The resulting allelic data were used to construct an unweighted pair group method using arithmetic averages (UPGMA) dendrogram and STRUCTURE diagram to elucidate relationships among the accessions. Results showed 11 consensus groups with EFB-resistant or tolerant accessions in all, providing strong evidence that EFB resistance is relatively widespread across the genus Corylus. Furthermore, open-pollinated seedlings tended to group together with reference accessions of similar geographic origins, providing insight into their genetic backgrounds. The results of this study add to the growing body of knowledge of hazelnut genetic resources and highlight recently introduced EFB-resistant seedling germplasm as new, unrelated genetic pools of resistance.

Anisogramma anomala, a biotrophic ascomycete in the order Diaporthales, causes eastern filbert blight (EFB) of hazelnuts (Corylus spp.). Until recently, little has been documented on its genetic diversity and population structure. In this study, 18 simple sequence repeat markers were used to fingerprint 182 accessions of the fungus originating from across North America. Our results, based on summary statistics of the allelic data, a discriminant analysis of principal components (DAPC) scatterplot, an unweighted pair group method with arithmetic mean (UPGMA) dendrogram, and analysis of multilocus genotypes, show that A. anomala exhibits considerable genetic diversity across multiple populations. Eleven clusters were resolved from the DAPC scatterplot, five of which were validated by statistically supported clusters in the UPGMA dendrogram. The 11 DAPC clusters were statistically significant via an analysis of molecular variance. Dendrogram topology and DAPC scatterplot groups showed some correlation with collection origin; samples collected in proximity tended to cluster together and be genetically similar. However, some locations held populations that were diverse and some populations with a high degree of similarity had disparate origins, suggesting movement by humans. Overall, the results demonstrate the presence of multiple, genetically distinct populations of A. anomala in North America and serve as a reference to assist in understanding and managing EFB.