Denita Hadziabdic

Genetic diversity of flowering dogwood in the Great Smoky Mountains National Park

In the past three decades, flowering dogwood (Cornus florida) populations have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48% to 98%, raising the concern that the genetic diversity of this native tree has been reduced significantly. In this study, we investigated levels of genetic diversity and population structure of flowering dogwood populations in the Great Smoky Mountains National Park (GSMNP). Understanding the factors influencing geographic distribution of genetic variation is one of the major concerns for preserving biodiversity and conservation of native populations. Eighteen microsatellite loci were used to evaluate the level and distribution of genetic variation of native flowering dogwood trees throughout the GSMNP. Significant genetic structure exists at both landscape and local levels. Two genetic clusters exist within the park and are separated by the main dividing ridges of the Great Smoky Mountains. The differentiation of the clusters is subtle, but statistically significant. Gene flow, evident through low-elevation corridors, indicates that nonrandom mating occurs between related individuals despite wide dispersal of seeds. Although high mortality rate and reduced fecundity caused by dogwood anthracnose severely affected native flowering dogwood populations throughout the entire GSMNP, this study confirmed that considerable genetic diversity still exists at the population level. It seems unlikely that recent demographic dynamics have resulted in a depletion of genetic variation.

Development and characterization of sixteen microsatellite loci for Geosmithia morbida, the causal agent of thousand canker disease in black walnut (Juglans nigra)

Sixteen polymorphic microsatellite loci were identified from the fungal pathogen Geosmithia morbida. Loci were characterized for 13 different isolates collected in 2010 from symptomatic black walnut trees in Tennessee. A total of 77 loci were tested and 16 of those were optimized, screened and selected for diversity studies of G. morbida. Number of alleles per locus ranged from 3 to 8. These microsatellite loci will be useful for rapid disease diagnostic, population genetic analyses on a global scale as well as further epidemiological studies of G. morbida.

Analysis of genetic diversity in flowering dogwood natural stands using microsatellites: the effects of dogwood anthracnose.

Flowering dogwood (Cornus florida L.) populations recently have experienced severe declines caused by dogwood anthracnose. Mortality has ranged from 48 to 98%, raising the concern that genetic diversity has been reduced significantly. Microsatellite data were used to evaluate the level and distribution of genetic variation throughout much of the native range of the tree. Genetic variation in areas affected by anthracnose was as high as or higher than areas without die-offs. We found evidence of four widespread, spatially contiguous genetic clusters. However, there was little relationship between geographic distance and genetic difference. These observations suggest that high dispersal rates and large effective population sizes have so far prevented rapid loss of genetic diversity. The effects of anthracnose on demography and community structure are likely to be far more consequential than short-term genetic effects.

Development and characterization of microsatellites for switchgrass rust fungus (Puccinia emaculata)

Ten polymorphic microsatellite loci from the fungus Puccinia emaculata, responsible for rust disease of switchgrass (Panicum virgatum) were developed. Loci were characterized using 20 single-pustule derived isolates of P. emaculata collected from switchgrass plants growing in the southeastern US. The number of alleles per locus ranged from 2 to 5. Observed heterozygosity ranged from 0 to 0.89 and expected heterozygosity ranged from 0.21 to 0.77. These microsatellite loci provide a sufficient set of markers to perform genetic diversity and epidemiological studies of P. emaculata.

Genetic differentiation and spatial structure of Geosmithia morbida, the causal agent of thousand cankers disease in black walnut (Juglans nigra).

The main objectives of this study were to evaluate genetic composition of Geosmithia morbida populations in the native range of black walnut and provide a better understanding regarding demography of the pathogen. The fungus G. morbida, and the walnut twig beetle, Pityophthorus juglandis, have been associated with a disease complex of black walnut (Juglans nigra) known as thousand cankers disease (TCD). The disease is manifested as branch dieback and canopy loss, eventually resulting in tree death. In 2010, the disease was detected in black walnut in Tennessee, and subsequently in Virginia and Pennsylvania in 2011 and North Carolina in 2012. These were the first incidences of TCD east of Colorado, where the disease has been established for more than a decade on indigenous walnut species. A genetic diversity and population structure study of 62 G. morbida isolates from Tennessee, Pennsylvania, North Carolina and Oregon was completed using 15 polymorphic microsatellite loci. The results revealed high haploid genetic diversity among seven G. morbida populations with evidence of gene flow, and significant differentiation among two identified genetic clusters. There was a significant correlation between geographic and genetic distance. Understanding the genetic composition and demography of G. morbida can provide valuable insight into recognizing factors affecting the persistence and spread of an invasive pathogen, disease progression, and future infestation predictions. Overall, these data support the hypotheses of two separate, highly diverse pathogen introductions into the native range of black walnut.

Analyzing Microsatellites Using the QIAxcel System

Microsatellites are ubiquitous throughout eukaryotic genomes and are useful in analyzing populations and genetic diversity. The QIAxcel system, an automated capillary electrophoresis device, allows the user to determine the size of microsatellite fragments, to discern allelic polymorphisms among individuals, and to differentiate homozygous and heterozygous individuals. This system provides comparable base pair resolution to more expensive systems at a relatively affordable cost.

Ten polymorphic microsatellite loci identified from a small insert genomic library for Peronospora tabacina

Ten polymorphic microsatellite loci for the obligate biotrophic, oomycete pathogen of tobacco, Peronospora tabacina, were identified from a small insert genomic library enriched for GT motifs. Eighty-five percent of the 162 loci identified were composed of dinucleotide repeats, whereas only 4% and 11% were tri-and tetra-nucleotide repeats respectively. About 82% of all the microsatellites were perfect and within the library; only about 7% of the loci were duplicated. Primers were designed for 63 loci; 10 loci were polymorphic, 19 were monomorphic and 34 either failed to amplify or produced ambiguous/inconsistent results. The 10 polymorphic loci were characterized with 44 isolates of P. tabacina collected from tobacco plants growing in Europe, the Near East and North and South America. The number of alleles per locus was either three or four with a mean of 3.2, and the mean number of genotypes per locus was 3.6. Observed heterozygosity was 0.32–0.95, whereas expected heterozygosity was 0.44–0.69 for these loci. All loci except PT054 did not conform to the Hardy-Weinberg distribution. Polymorphic information content (PIC) for the loci was 0.35–0.69 with a mean of 0.50. These microsatellite loci provide a set of markers sufficient to perform genetic diversity and population studies of P. tabacina, and possibly other species of Peronospora.

Development of microsatellite loci in Pityophthorus juglandis, a vector of thousand cankers disease in Juglans spp.

Using next-generation sequencing, 18 microsatellite loci were developed and characterized for walnut twig beetle, Pityophthorus juglandis, a vector of thousand cankers disease (TCD) affecting Juglans spp. Although all Juglans species are susceptible to TCD infection, native populations of J. nigra and J. cinerea, which is endangered in Canada, are most susceptible and threatened by habitat loss. Novel primers amplified di-, tri-, and tetra nucleotide repeats and detected 4–14 alleles per locus. Averaged observed and expected heterozygosity was 0.22 and 0.67, respectively. Our results indicate that P. juglandis microsatellite loci can be used to investigate genetic diversity and population structure of this vector across a widespread geography. These markers will be useful tools for evaluating genetic structure of P. juglandis population outbreaks and developing appropriate conservation strategies. Microsatellite loci obtained in this study can also be utilized to determine relationships of P. juglandis to other closely related Pityophthorus spp.

Confirmation of independent introductions of an exotic plant pathogen of Cornus species, Discula destructiva, on the east and west coasts of North America.

Cornus florida (flowering dogwood) and C. nuttallii (Pacific dogwood) are North American native tree species that belong to the big-bracted group of dogwoods. Cornus species are highly valued for their ornamental characteristics, and have fruits that contain high fat content for animals. Also, they are an important understory tree in natural forests. Dogwood anthracnose, caused by Discula destructiva, was observed in the late 1970s on the east and west coasts of the United States and by 1991 had quickly spread throughout most of the native ranges of C. florida and C. nuttalli. We investigated the genetic diversity and population structure of 93 D. destructiva isolates using 47 microsatellite loci developed from the sequenced genome of the type strain of D. destructiva. Clone-corrected data indicated low genetic diversity and the presence of four genetic clusters that corresponded to two major geographic areas, the eastern United States and the Pacific Northwest, and to the two collection time periods when the isolates were collected (pre- and post-1993). Linkage disequilibrium was present in five out of six subpopulations, suggesting that the fungus only reproduced asexually. Evidence of population bottlenecks was indicated across four identified genetic clusters, and was probably the result of the limited number of founding individuals on both coasts. These results support the hypothesis that D. destructiva is an exotic pathogen with independent introductions on the east and west coasts of North America. We also tested the cross-amplification of these microsatellite primers to other Discula species. Genomic DNA from 17 isolates of four other Discula species and two isolates of Juglanconis species (formerly Melanconis species) were amplified by 17 of 47 primer pairs. These primers may be useful for investigating the genetic diversity and population structure of these Discula species.

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.