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Amnon Levi, Karen R. Harris-Shultz and Kai-shu Ling

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Karen R. Harris-Shultz, Brian M. Schwartz and Jeff A. Brady

The release of the bermudagrass (Cynodon spp.) triploid hybrid ‘Tifgreen’ revolutionized southeastern U.S. golf course greens. Off-types within this cultivar began to be identified soon after the initial plantings, and through the last 50 years, many of the best performing off-types have been released as new cultivars. Examination of some of the most popular somatic mutants with a new set of 47 simple sequence repeat (SSR) markers and 23 previously discovered genomic SSR markers identified five polymorphic fragments (as compared with ‘Tifgreen’) among three cultivars, TifEagle, MiniVerde, and Tifdwarf. Each polymorphism appears to be a slight increase/decrease in microsatellite repeat number and the polymorphic fragments are unique for each cultivar. Two polymorphic fragments were identified that were unique to ‘Tifdwarf’, one polymorphic fragment was unique to ‘TifEagle’, and two polymorphic fragments were unique to ‘MiniVerde’. Furthermore, three of the five polymorphic markers display an additional allele only in the shoot tissue but not in the root tissue of ‘TifEagle’ and ‘Tifdwarf’. This finding suggests that ‘TifEagle’ and ‘Tifdwarf’ are somatic chimeras. This set of SSR markers identifies repeatable polymorphic fragments among multiple ‘Tifgreen’-derived cultivars and gives insight into the nature of the mutations that exist within ‘Tifgreen’.

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Karen R. Harris, W. Patrick Wechter and Amnon Levi

Sixty-six watermelon (Citrullus lanatus var. lanatus) disease resistance gene analogs were cloned from ‘Calhoun Gray’, PI 296341, and PI 595203 using degenerate primers to select for the nucleotide binding sites (NBS) from the NBS–leucine-rich repeat (LRR) resistance gene family. After contig assembly, watermelon resistance gene analogs (WRGA) were identified and amino acid sequence alignment revealed that these groups contained motifs characteristic of NBS-LRR resistance genes. Using cluster analysis, eight groups of WRGA were identified and further characterized as having homology to Drosophila Toll and mammalian interleukin-1 receptor (TIR) and non-TIR domains. Three of these WRGA as well as three disease-related watermelon expressed sequence tag homologs were placed on a test-cross map. Linkage mapping placed the WRGA on linkage group XIII, an area on the watermelon map where resistance gene analogs cluster. In addition, these WRGA sequence-tagged sites (STS) were amplified from various genera of the Cucurbitaceae indicating that conservation of resistance gene analogs exists among cucurbits. These WRGA-STS markers may be useful in marker-assisted selection for the improvement for disease resistance in watermelon.

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Amnon Levi, William P. Wechter, Karen R. Harris, Angela R. Davis and Zhangjun Fei

In this study, we report a simple procedure for developing and using new types of polymerase chain reaction (PCR) primers, named “high-frequency oligonucleotides–targeting active genes” (HFO-TAG). The HFO-TAG primers were constructed by first using a “practical extraction and report language” script to identify oligonucleotides (8, 9, and 10 bases) that exist in high frequency in 4700 expressed sequence tag (EST)-unigenes of watermelon (Citrullus lanatus) fruit. This computer-based screening yielded 3162 oligonucleotides that exist 32 to 335 times in the 4700 EST-unigenes. Of these, 192 HFO-TAG primers (found 51 to 269 times in the 4700 EST-unigenes) were used to amplify genomic DNA of four closely related watermelon cultivars (Allsweet, Crimson Sweet, Charleston Gray, and Dixielee). The average number of DNA fragments produced by a single HFO-TAG primer among these four watermelon cultivars was considerably higher (an average of 5.74 bands per primer) than the number of fragments produced by intersimple sequence repeat (ISSR) or randomly amplified polymorphic DNA (RAPD) primers (an average of 2.32 or 4.15 bands per primer, respectively). The HFO-TAG primers produced a higher number of polymorphic fragments (an average of 1.77 polymorphic fragments per primer) compared with the ISSR and RAPD primers (an average of 0.89 and 0.47 polymorphic fragments per primer, respectively). Amplification of genomic DNA from 12 watermelon cultivars and two U.S. Plant Introductions with the HFO-TAG primers produced a significantly higher number of fragments than RAPD primers. Also, in PCR experiments examining the ability of primers to amplify fragments from a watermelon cDNA library, the HFO-TAG primers produced considerably more fragments (an average of 6.44 fragments per primer) compared with ISSR and RAPD primers (an average of 3.59 and 2.49 fragments per primer, respectively). These results indicate that the HFO-TAG primers should be more effective than ISSR or RAPD primers in targeting active gene loci. The extensive EST database available for a large number of plant and animal species should be a useful source for developing HFO-TAG primers that can be used in genetic mapping and phylogenic studies of important crop plants and animal species.

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Karen R. Harris-Shultz, Brian M. Schwartz, Wayne W. Hanna and Jeff A. Brady

Genetic linkage maps of bermudagrass (Cynodon spp.) species using 118 triploid individuals derived from a cross of T89 [C. dactylon (2n = 4x = 36)] and T574 [C. transvaalensis (2n = 2x = 18)] were enriched with expressed sequence tags-derived simple sequence repeat (EST-SSR) markers. Primers were developed from 53 ESTs containing SSRs producing 75 segregating markers from which 28 could be mapped to the T89 and T574 genetic maps. With the addition of previously generated marker data, 26 T89 linkage groups and eight T574 linkage groups were formed using a log-of-odds (LOD) value of 4.0. The T89 and T574 linkage maps spanned 1055 cM and 311.1 cM and include 125 and 36 single-dose amplified fragments (SDAFs), respectively. Many of the SDAFs displayed disomic segregation and thus T89 may be a segmental allotetraploid or an allotetraploid. The additional EST-SSR markers add value to the maps by increasing marker density and provide markers that can be easily transferred to other bermudagrass populations. Furthermore, EST-SSRs can be immediately used to assess genetic diversity, identify non-mutated cultivars of bermudagrass, confirm pedigrees, and differentiate contaminants from cultivars derived from ‘Tifgreen’.

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Karen R. Harris, Brian M. Schwartz, Andrew H. Paterson and Jeff A. Brady

Thirty-one partial bermudagrass (Cynodon spp.) disease-resistance gene analogs (BRGA) were cloned and sequenced from diploid, triploid, tetraploid, and hexaploid bermudagrass using degenerate primers to target the nucleotide binding site (NBS) of the NBS–leucine-rich repeat (LRR) resistance gene family. Alignment of deduced amino acid sequences revealed that the conserved motifs of the NBS are present and all sequences have non-Drosophila melanogaster Toll and mammalian interleukin-1 receptor (TIR) motifs. Using a neighbor-joining algorithm, a dendrogram was created and nine groups of deduced amino acid sequences from bermudagrass could be identified from those sequences that span the NBS. Four BRGA markers and 15 bermudagrass expressed sequence tags (ESTs) with similarity to resistance genes or resistance gene analogs were placed on a bermudagrass genetic map. Multiple BRGA and EST markers mapped on T89 linkage groups 1a and 5a and clusters were seen on T89 19 and two linkage groups previously unidentified. In addition, three primers made from BRGA groups and ESTs with similarity to NBS-LRR resistance genes amplify NBS-LRR analogs in zoysiagrass (Zoysia japonica or Z. matrella) or seashore paspalum (Paspalum vaginatum). This gives evidence of conservation of NBS-LRR analogs among the subfamilies Chloridoideae and Panicoideae. Once disease resistance genes are identified, these BRGA and EST markers may be useful in marker-assisted selection for the improvement of disease resistance in bermudagrass.

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Karen R. Harris, Kai-Shu Ling, W. Patrick Wechter and Amnon Levi

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses affecting watermelon [Citrullus lanatus (Thunb.) Matsun & Nakai var. lanatus] in the United States. The ZYMV-Florida strain (ZYMV-FL) is considered a major limitation to commercial watermelon production in the United States. Inheritance of resistance to ZYMV-FL is conferred by a recessive gene. This report describes the identification of single-reaction, polymerase chain reaction-based markers linked to the ZYMV-FL resistance gene in watermelon. In this study, we identified a marker ZYMV-resistant polymorphism (ZYRP) linked to the ZYMV-FL resistance gene locus (genetic distance of 8 cM) in an F2 population, and in a backcross one to the resistant parent population (BC1R) (genetic distance of 13 cM). The identification of a single nucleotide polymorphism within the ZYRP marker for the parental genotypes allowed the development of a sequence-characterized amplification region marker linked to the ZYMV-FL resistance gene. Experiments using a BC2F2 population derived from the U.S. Plant Introduction 595203 (C. lanatus var. lanatus) and the recurrent parent ‘Charleston Gray’ indicated that the ZYRP marker can be used in marker-assisted selection to identify genotypes containing the gene conferring ZYMV-FL resistance in watermelon.

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Clinton J. Steketee, Alfredo D. Martinez-Espinoza, Karen R. Harris-Shultz, Gerald M. Henry and Paul L. Raymer

Seashore paspalum (Paspalum vaginatum Swartz) is a warm-season turfgrass species primarily used on golf courses and athletic fields, and is often impacted by the disease dollar spot caused by Sclerotinia homoeocarpa F.T. Bennett. Dollar spot is the most common and economically important turfgrass disease in North America, and current management of this disease relies heavily on frequent fungicide applications. An alternate management strategy is host plant resistance, but a better understanding of the interactions between pathogen isolates and the host species is needed to effectively incorporate this resistance into elite seashore paspalum genotypes. The goal of this study was to gather host plant/isolate response data that could be used to develop an effective and efficient screening protocol for resistance to this important disease. Five genotypes of seashore paspalum (‘Aloha’, ‘SeaIsle 2000’, ‘SeaIsle 1’, ‘SeaIsle Supreme’, and 05-1743) varying in dollar spot resistance were inoculated with five isolates of S. homoeocarpa in repeated field studies during 2012 and 2013. Isolates used were from three warm-season and one cool-season turfgrass species. Inoculated plots were evaluated visually and using digital image analysis (DIA) for disease development over time and for number and area of infection centers at two rating dates each year. Statistical differences among the seashore paspalum genotypes and inoculation/isolate treatments were detected for area under the disease progress curve (AUDPC) values, number of infection centers, and infection center area. A significant interaction between seashore paspalum genotype and S. homoeocarpa isolate effects was not observed, indicating that host plant resistance genes are likely not isolate specific. Using this information, breeders should be able to use one highly virulent S. homoeocarpa isolate to screen for host plant resistance in seashore paspalum.

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Brian M. Schwartz, Ryan N. Contreras, Karen R. Harris-Shultz, Douglas L. Heckart, Jason B. Peake and Paul L. Raymer

Seashore paspalum is a salt tolerant, predominately diploid (2n = 2x = 20) species that is well adapted to coastal regions in tropical and subtropical environments. Because a majority of the available cultivars are propagated vegetatively and most genotypes are cross-fertile, a sterile cultivar that does not produce segregating seedlings would benefit sod growers and turfgrass managers who demand uniformity for certification and performance. Therefore, an experiment was conducted to create a colchicine-induced polyploid seashore paspalum. One triploid (2n = 3x = 30) genotype (11-TSP-1) was identified and remains stable. Although there is a possibility that this event was triggered by the colchicine treatment, a more likely explanation is that it resulted from the union of a reduced and an unreduced gamete. Pollen shed was observed from 11-TSP-1 in 2011, but individual pollen grains stained with iodine–potassium iodide were irregularly shaped and typically had lower starch content than pollen from several diploid cultivars. The leaf width of 11-TSP-1 was statistically equal to that of the seashore paspalum cultivar SeaStar, indicating its potential for use as a fine turf. 11-TSP-1 had both superior visual color and a dark green color index when compared with ‘SeaStar’. Future study of the reproductive fertility and more extensive field testing of this genotype should be carried out to determine its turfgrass potential. Chemical names used: 4′, 6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), iodine-potassium iodide (I2-KI), propidium iodide (PI).

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Karen R. Harris-Shultz, Susana Milla-Lewis, Aaron J. Patton, Kevin Kenworthy, Ambika Chandra, F. Clint Waltz, George L. Hodnett and David M. Stelly

Zoysiagrass (Zoysia sp.) is used as a warm-season turfgrass for lawns, parks, and golf courses in the warm, humid and transitional climatic regions of the United States. Zoysiagrass is an allotetraploid species (2n = 4x = 40) and some cultivars are known to easily self- and cross-pollinate. Previous studies showed that genetic variability in the clonal cultivars Emerald and Diamond was likely the result of contamination (seed production or mechanical transfer) or mislabeling. To determine the extent of genetic variability of vegetatively propagated zoysiagrass cultivars, samples were collected from six commercially available zoysiagrass cultivars (Diamond, Emerald, Empire, JaMur, Meyer, Zeon) from five states (Arkansas, Florida, Georgia, North Carolina, Texas). Two of the newest cultivar releases (Geo and Atlantic) were to serve as outgroups. Where available, one sample from university research plots and two samples from sod farms were collected for each cultivar per state. Forty zoysiagrass simple sequence repeat (SSR) markers and flow cytometry were used to compare genetic and ploidy variation of each collected sample to a reference sample. Seventy-five samples were genotyped and an unweighted pair group method with arithmetic mean clustering revealed four groups. Group I (Z. japonica) included samples of ‘Meyer’ and Empire11 (‘Empire’ sample at location #11), Group II (Z. japonica × Z. pacifica) included samples of ‘Emerald’ and ‘Geo’, Group III (Z. matrella) included samples of ‘Diamond’ and ‘Zeon’, and Group IV (Z. japonica) consisted of samples from ‘Empire’, ‘JaMur’, ‘Atlantic’, and Meyer3 (‘Meyer’ at sample location #3). Samples of ‘Empire’, ‘Atlantic’, and ‘JaMur’ were indistinguishable with the markers used. Four samples were found to have alleles different from the respective reference cultivar, including two samples of ‘Meyer’, one sample of ‘Empire’, and one sample of ‘Emerald’. Three of these samples were from Texas and one of these samples was from Florida. Three of the four samples that were different from the reference cultivar were university samples. In addition, one sample, Empire11, was found to be an octoploid (2n = 8x = 80). For those samples that had a fingerprint different from the reference cultivar, contamination, selfing, and/or hybridization with other zoysiagrasses may have occurred.