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Kentucky bluegrass (Poa pratensis L.) is an important perennial forage and amenity turfgrass species used throughout temperate regions in North America and Europe. Kentucky bluegrass cultivars and accessions exhibit a wide range of ploidy levels, morphological traits, turfgrass quality characteristics, and tolerances to both biotic and abiotic stresses. As such, cultivar classification systems have been developed to categorize Kentucky bluegrass cultivars and accessions on the basis of pedigree, turf performance, morphological traits, and genetic relatedness (DNA markers). The objectives of the current study were to assess the genetic diversity among and within Kentucky bluegrass entries using simple sequence repeat (SSR) markers, and to categorize new germplasm in the Kentucky bluegrass classification system. In this study, 21 SSR markers were used to genotype eight individuals from each of 144 bluegrass cultivars and accessions (1152 total samples). The SSR markers successfully categorized all entries into distinct classification groups and provided justification for a partial revision to the previous DNA marker-based classification system. The majority of cultivars and accessions were uniquely identified with the current set of SSR markers, and SSR marker-based genetic relationships of individuals within classification types showed agreement with breeding history records. Over 60 new Kentucky bluegrass National Turfgrass Evaluation Program entries were placed into updated classification groups, and the SSR markers used in this study can be used in the future to genotype and assign new cultivars and accessions into Kentucky bluegrass classification types and assess the genetic relatedness among entries.

Leveraging the heightened levels of polymorphism in NB-ARC-related protein encoding genes in higher plants, a bioinformatic pipeline was created to identify regions in this gene family from sequenced plant genomes that exhibit fragment length or single nucleotide differences in different accessions of the same species. Testing this approach with the aquatic plant Spirodela polyrhiza demonstrated its superior performance in comparison with currently available genotyping technologies based on PCR amplification. Rapid and economical genotyping tools that can reliably distinguish species and intraspecific variations in plants can be powerful tools for biogeographical and ecological studies. Clones of the cosmopolitan duckweed species, Spirodela polyrhiza, are difficult to distinguish morphologically due to their highly abbreviated architecture and inherently low levels of sequence variation. The use of plastidic markers and generic Amplification Fragment Length Polymorphism approaches have met with limited success in resolving clones of S. polyrhiza from diverse geographical locales. Using whole genome sequencing data from nine S. polyrhiza clones as a training set, we created an informatic pipeline to identify and rank polymorphic regions from nuclear-encoded NB-ARC-related genes to design markers for PCR, Sanger sequencing (barcoding), and fragment length analysis. With seven primer sets, we found 21 unique fingerprints from a set of 23 S. polyrhiza clones. However, three of these clones share the same fingerprint and are indistinguishable by these markers. These primer sets can also be used as interspecific barcoding tools to rapidly resolve S. polyrhiza from the closely related S. intermedia species without the need for DNA sequencing. Our work demonstrates a general approach of using hyper-polymorphic loci within genomes as a resource to produce facile tools that can have high resolving power for genotyping applications.

Genetic relationships among Agrostis species used for turf have been difficult to discern. Recent studies have either confirmed or contradicted previously proposed genetic relationships based on chromosome pairing behavior of inter-specific hybrids. The objective of the current study was to assess genetic relationships among Agrostis cultivars and accessions by using newly available A. stolonifera microsatellite (SSR) markers. Nuclear SSR (nuSSR) and chloroplast SSR (cpSSR) markers were used to genotype 16 individuals from each of 74 Agrostis cultivars and accessions. Genetic relationships based on nuSSR markers most closely resembled species relationships proposed by Jones in the 1950s. Contrary to the work of Jones, genetic relationships based on cpSSR markers indicated that A. canina was more closely related to A. stolonifera than to A. capillaris. We hypothesize that chloroplast introgression via interspecific hybridization between A. canina and A. stolonifera resulted in these species sharing common chloroplast genome lineages, while maintaining disparate nuclear genome lineages. Genetic relationships within Agrostis species based on nuSSR markers closely matched known pedigree relationships. Bayesian clustering analysis of nuSSR markers indicated that most modern seeded A. stolonifera cultivars exhibited high levels of admixture. Our study confirms that nuSSR markers distinguish Agrostis species and cultivars, and are valuable for studying genetic diversity and genetic relationships within the genus Agrostis.

Blueberry anthracnose, caused by Colletotrichum fioriniae, is a pre- and postharvest disease of cultivated highbush blueberry (Vaccinium corymbosum). During disease development, the pathogen undergoes several lifestyle changes during host colonization, including epiphytic, quiescent, and necrotrophic phases. It is not clear, however, what if any host signals alter the pattern of colonization during the initial epiphytic phase and infection. This research investigated the role of blueberry floral extracts (FE) on fungal development. Results show that FE significantly increased both the quantity and rate of secondary conidiation and appressorial formation in vitro, suggesting that floral components could decrease the minimum time required for infection. Activity of FE was readily detected in water collected from field samples, where secondary conidiation and appressorial formation decreased as rainwater collections were further removed from flowers. A comparison of FE from four blueberry cultivars with different levels of field susceptibility revealed that appressorial formation but not secondary conidiation significantly increased with the FE from susceptible cultivars versus resistant cultivars. Inoculum supplemented with FE produced higher levels of disease on ripe blueberry fruit as compared with inoculum with water only. Flowers from other ericaceous species were found to also induce secondary conidiation and appressorial formation of C. fioriniae. This research provides strong evidence that flowers can contribute substantially to the infection process of C. fioriniae, signifying the importance of the bloom period for developing effective disease management strategies.

Limited understanding of sweet basil (Ocimum basilicum L.) genetics and genome structure has reduced efficiency of breeding strategies. This is evidenced by the rapid, worldwide dissemination of basil downy mildew (Peronospora belbahrii) in the absence of resistant cultivars. In an effort to improve available genetic resources, expressed sequence tag simple sequence repeat (EST-SSR) and single nucleotide polymorphism (SNP) markers were developed and used to genotype the MRI x SB22 F2 mapping population, which segregates for response to downy mildew. SNP markers were generated from genomic sequences derived from double digestion restriction site associated DNA sequencing (ddRADseq). Disomic segregation was observed in both SNP and EST-SSR markers providing evidence of an O. basilicum allotetraploid genome structure and allowing for subsequent analysis of the mapping population as a diploid intercross. A dense linkage map was constructed using 42 EST-SSR and 1,847 SNP markers spanning 3,030.9 cM. Multiple quantitative trait loci (QTL) model (MQM) analysis identified three QTL that explained 37-55% of phenotypic variance associated with downy mildew response across three environments. A single major QTL, dm11.1 explained 21-28% of phenotypic variance and demonstrated dominant gene action. Two minor QTL dm9.1 and dm14.1 explained 5-16% and 4-18% of phenotypic variance, respectively. Evidence is provided for an additive effect between the two minor QTL and the major QTL dm11.1 increasing downy mildew susceptibility. Results indicate that ddRADseq-facilitated SNP and SSR marker genotyping is an effective approach for mapping the sweet basil genome.

In New Jersey, Phytophthora cinnamomi is the pathogen most commonly isolated from diseased roots and runners of the cultivated cranberry (Vaccinium macrocarpon). A second distinct species of Phytophthora has been isolated from dying cranberry plants and surface irrigation water. This species is homothallic with paragynous antheridia and ellipsoid-limoniform, nonpapillate sporangia. It was tentatively identified as P. megasperma in an earlier report. Laboratory experiments demonstrate that the cardinal temperatures for vegetative growth are between 5 and 30 degrees C with an optimum near 25 degrees C. Sporangia are produced at temperatures between 10 and 20 degrees C with the majority of sporangia produced at 10 and 15 degrees C. In pathogenicity tests, no growth effect was observed on cranberry plants (cv. Early Black) when tests were conducted at 25 degrees C; however, significant reductions in plant growth occurred when tests were conducted at 15 degrees C. This species was insensitive to metalaxyl but was sensitive to buffered phosphorous acid. Sequence analysis of the internal transcribed spacer 1 (ITS1), 5.8S rDNA, and ITS2 regions place these isolates in Phytophthora clade 6 with greatest similarity to Phytophthora taxon raspberry. To our knowledge, this is the first report of isolates of this affiliation in North America. However, the observation of low temperature preferences makes this species unique in an otherwise high temperature clade. The isolates described in this study are tentatively classified as Phytophthora taxon cranberry.