Mark V. Coggeshall

Susceptibility of Walnut and Hickory Species to Geosmithia morbida

Thousand cankers disease (TCD) of walnut is a result of feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, and subsequent canker formation caused by Geosmithia morbida around galleries. TCD has caused extensive morbidity and mortality to Juglans nigra in the western United States and, in 2010, was discovered in the eastern United States, where the tree is a highly valuable timber resource. WTB and G. morbida also have been found in J. regia orchards throughout major production areas in California, and the numbers of damaged trees are increasing. We tested the susceptibility of walnut and hickory species to G. morbida in greenhouse and field studies. Carya illinoinensis, C. aquatica, and C. ovata were immune. All walnut species tested, including J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. microcarpa, J. nigra, and J. regia, developed cankers following inoculation with G. morbida. J. nigra was the most susceptible, whereas J. major, a native host of the WTB and, presumably, G. morbida, had smaller and more superficial cankers. Canker formation differed among maternal half-sibling families of J. nigra and J. cinerea, indicating genetic variability in resistance to G. morbida. Our inoculation studies with G. morbida have corroborated many of the field observations on susceptibility of walnut and hickory species to TCD, although the ability of the WTB to successfully attack and breed in walnut is also an important component in TCD resistance.

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.

Development of Genomic Microsatellites in Gleditsia triacanthos (Fabaceae) Using Illumina Sequencing

Premise of the study: Fourteen genomic microsatellite markers were developed and characterized in honey locust, Gleditsia triacanthos, using Illumina sequencing. Due to their high variability, these markers can be applied in analyses of genetic diversity and structure, and in mating system and gene flow studies. Methods and Results: Thirty-six individuals from across the species range were included in a genetic diversity analysis and yielded three to 20 alleles per locus. Observed heterozygosity and expected heterozygosity ranged from 0.214 to 0.944 and from 0.400 to 0.934, respectively, with minimal occurrence of null alleles. Regular segregation of maternal alleles was observed at seven loci and moderate segregation distortion at four of 11 loci that were heterozygous in the seed parent. Conclusions: Honey locust is an important agroforestry tree capable of very fast growth and tolerance of poor site conditions. This is the first report of genomic microsatellites for this species.

EST-SSR markers reveal synonymies, homonymies and relationships inconsistent with putative pedigrees in chestnut cultivars

Over the last two centuries, chestnut breeding programs in Europe and Asia have generated an array of chestnut interspecific hybrids, primarily of European (Castanea sativa), Japanese (C. crenata) and Chinese (C. mollissima) ancestry. During this same period, Europeans colonizing North America imported hybrid chestnuts and made interspecific hybrids with native chestnuts, primarily American chestnut (C. dentata). The importation of Chinese chestnut into the United States in the late 19th century also introduced chestnut blight, which triggered an additional interspecific hybridization effort in an attempt to develop blight resistant American chestnuts. Chestnut cultivars used for nut production in the United States and Canada have arisen against this background of non-native introductions and extensive hybridizing. The development of regionally adapted nut producing trees with dependable crops of high quality nuts requires sorting out the identities of existing cultivars. We chose 11 EST-SSR markers from C. mollissima for the initial task of genotyping 65 chestnut cultivars that grow well in the central United States. Many of these cultivars have interspecific pedigrees involving two or more species. We found extensive homonymies and synonymies, genetic groups inconsistent with published pedigrees, contradictory pedigrees and evidence for incorrect species assignments. Accurate inference of the interspecific ancestries of cultivars grown in the United States and Canada will require genotyping of species reference sets for C. sativa, C. crenata, C. mollissima, C. dentata and possibly C. pumila (the Ozark and Allegheny chinquapins).

High-quality genetic mapping with ddRADseq in the non-model tree Quercus rubra

BackgroundRestriction site associated DNA sequencing (RADseq) has the potential to be a broadly applicable, low-cost approach for high-quality genetic linkage mapping in forest trees lacking a reference genome. The statistical inference of linear order must be as accurate as possible for the correct ordering of sequence scaffolds and contigs to chromosomal locations. Accurate maps also facilitate the discovery of chromosome segments containing allelic variants conferring resistance to the biotic and abiotic stresses that threaten forest trees worldwide. We used ddRADseq for genetic mapping in the tree Quercus rubra, with an approach optimized to produce a high-quality map. Our study design also enabled us to model the results we would have obtained with less depth of coverage.ResultsOur sequencing design produced a high sequencing depth in the parents (248×) and a moderate sequencing depth (15×) in the progeny. The digital normalization method of generating a de novo reference and the SAMtools SNP variant caller yielded the most SNP calls (78,725). The major drivers of map inflation were multiple SNPs located within the same sequence (77% of SNPs called). The highest quality map was generated with a low level of missing data (5%) and a genome-wide threshold of 0.025 for deviation from Mendelian expectation. The final map included 849 SNP markers (1.8% of the 78,725 SNPs called). Downsampling the individual FASTQ files to model lower depth of coverage revealed that sequencing the progeny using 96 samples per lane would have yielded too few SNP markers to generate a map, even if we had sequenced the parents at depth 248×.ConclusionsThe ddRADseq technology produced enough high-quality SNP markers to make a moderately dense, high-quality map. The success of this project was due to high depth of coverage of the parents, moderate depth of coverage of the progeny, a good framework map, an optimized bioinformatics pipeline, and rigorous premapping filters. The ddRADseq approach is useful for the construction of high-quality genetic maps in organisms lacking a reference genome if the parents and progeny are sequenced at sufficient depth. Technical improvements in reduced representation sequencing (RRS) approaches are needed to reduce the amount of missing data.

A Novel Molecular Toolkit for Rapid Detection of the Pathogen and Primary Vector of Thousand Cankers Disease

Thousand Cankers Disease (TCD) of Juglans and Pterocarya (Juglandaceae) involves a fungal pathogen, Geosmithia morbida, and a primary insect vector, Pityophthorus juglandis. TCD was described originally from dying Juglans nigra trees in the western United States (USA), but it was reported subsequently from the eastern USA and northern Italy. The disease is often difficult to diagnose due to the absence of symptoms or signs on the bark surface of the host. Furthermore, disease symptoms can be confused with those caused by other biotic and abiotic agents. Thus, there is a critical need for a method for rapid detection of the pathogen and vector of TCD. Using species-specific microsatellite DNA markers, we developed a molecular protocol for the detection of G. morbida and P. juglandis. To demonstrate the utility of the method for delineating TCD quarantine zones, we tested whether geographical occurrence of symptoms and signs of TCD was correlated with molecular evidence for the presence of the cryptic TCD organisms. A total of 1600 drill cores were taken from branch sections collected from three regions (n = 40 trees for each location): California-J. hindsii (heavy disease incidence); Tennessee-J. nigra (mild disease incidence); and outside the known TCD zone (Missouri-J. nigra, no record of the disease). California samples had the highest incidence of the TCD organisms (85%, 34/40). Tennessee had intermediate incidence (42.5%, 17/40), whereas neither organism was detected in samples from Missouri. The low cost molecular protocol developed here has a high degree of sensitivity and specificity, and it significantly reduces sample-processing time, making the protocol a powerful tool for rapid detection of TCD.

The first genetic linkage map for Fraxinus pennsylvanica and syntenic relationships with four related species

Green ash (Fraxinus pennsylvanica) is an outcrossing, diploid (2n=46) hardwood tree species, native to North America. Native ash species in North America are being threatened by the rapid invasion of emerald ash borer (EAB, Agrilus planipennis) from Asia. Green ash, the most widely distributed ash species, is severely affected by EAB infestation, yet few resources for genetic studies and improvement of green ash are available. In this study, a total of 5,712 high quality single nucleotide polymorphisms (SNPs) were discovered using a minimum allele frequency of 1% across the entire genome through genotyping-by-sequencing. We also screened hundreds of genomic- and EST-based microsatellite markers (SSRs) from previous de novo assemblies (Staton et al. 2015; Lane et al. 2016). A first genetic linkage map of green ash was constructed from 91 individuals in a full-sib family, combining 2,719 SNP and 84 SSR segregating markers among the parental maps. The consensus SNP and SSR map contains a total of 1,201 markers in 23 linkage groups spanning 2008.87cM, at an average inter-marker distance of 1.67 cM with a minimum logarithm of odds (LOD) of 6 and maximum recombination fraction of 0.40. Comparisons of the organization the green ash map with the genomes of asterid species coffee and tomato, and genomes of the rosid species poplar and peach, showed areas of conserved gene order, with overall synteny strongest with coffee.