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  • Author or Editor: Amnon Levi. x
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Previous studies identified three major chilling-responsive proteins of 65, 60, and 14 kDa whose levels increase in floral buds of blueberry during cold acclimation and decrease during deacclimation and resumption of growth. Characterization of these proteins found them to be members of a family of proteins responsive to drought and low temperature stress called dehydrins. The 65- and 60-kDa proteins were purified, digested into peptides, and several peptides from each were sequenced. The sequence information was used to synthesize degenerate DNA primers for amplification of a part of the gene(s) encoding these proteins. One pair of primers amplified a 200-bp fragment, which now has been cloned and sequenced. Within the 200-bp sequence is a motif conserved amongst dehydrins. Hybridization of the 200-bp fragment to RNA blots revealed homology to two chilling-responsive messages of 3.7 and 1.6 kb. The 200-bp fragment currently is being used to screen a cDNA library (prepared from RNA from cold acclimated blueberry floral buds) to isolate the full length cDNA clone.

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The coordinate expression of mRNA classes in pecan (Carya illinoensis) zygotic and somatic embryos has been studied. MRNA was isolated from zygotic embryos at early and late maturation stages (12 to 22 weeks post-pollination) and during germination. Additionally, mRNA was isolated from somatic embryos derived from a repetitive embryogenic system prior and after cold (6 weeks at 4°C) and desiccation treatments (5 days). These treatments have been determined to enhance somatic embryo conversion. The abundance of embryogenic mRNA classes was determined using various cloned cotton mRNA probes (Hughes and Galau, 1989). This study is a part of our efforts to elucidate the developmental and physiological differences between zygotic and somatic embryo systems in pecan.

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Repetitive somatic embryogenic lines of pecan (Carya illinoensis) were obtained and subcultured on basal WPM, following a one week induction of zygotic embryo tissue on modified WPM with 6 mg/L NAA. Gene expression of somatic embryos has been studied and compared with that occurring in zygotic embryos. Somatic embryos simultaneously expressed mRNA classes that are specific to each of the zygotic embryo cotyledon (Cot), maturation (Mat), and post abscission stages (Late embryogenesis, Lea). Somatic embryos exhibiting such multiple, nonregulated gene expression patterns have a low germination rate. Treatments found to enhance embryo germination (cold and desiccation) may be effective in part, by modifying gene expression patterns. Some of the Cot and Mat mRNA classes decreased following such treatments, while Lea mRNAs were not effected. Cold and desiccation treatments appear to coordinate gene expression in pecan somatic embryos, which might be associated with embryo germination.

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Southern root-knot nematodes (Meloidogyne incognita) are an important re-emerging pest of watermelon in the United States and worldwide. The re-emergence of root-knot nematodes (RKNs) in watermelon and other cucurbits is largely the result of the intensive cultivation of vegetable crops on limited agricultural lands coupled with the loss of methyl bromide for pre-plant soil fumigation, which has been the primary method for control of RKNs and many soilborne diseases of cucurbits and other vegetable crops for several decades. One alternative for managing RKN in watermelon is the use of resistant rootstocks for grafted watermelon. We have developed several RKN-resistant Citrullus lanatus var. citroides lines (designated RKVL for Root-Knot Vegetable Laboratory), which have shown promise as rootstocks for grafted watermelon. In 2011 and 2012, we demonstrated that F1 hybrids derived from our selected RKVL lines exhibited resistance to RKN that was equal to or greater than that of the parental RKVL lines when grown in fields highly infested with M. incognita. In 2011, although significant differences were not observed among rootstocks, the F1 hybrids produced slightly higher yields compared with the selected parental lines. Among the selected parental lines, RKVL 318 produced high yields in both years. In 2011, three of four RKVL parental lines and all four of their F1 hybrids produced greater (P < 0.05) fruit yields than self-grafted ‘Tri-X 313’, ‘Emphasis’ bottle gourd, and ‘Strong Tosa’ squash hybrid. In 2012, three RKVL F1 hybrid lines produced higher yields than the selected parents. Overall, these F1 hybrids were vigorous and should provide useful genetic material for selection and development of robust RKN-resistant C. lanatus var. citroides rootstock lines.

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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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Genetic relatedness was estimated among 42 U.S. plant introduction (PI) accessions of the genus Citrullus (37 PIs of which were reported to have disease resistance and five watermelon cultivars) using 30 RAPD primers. These primers produced 662 RAPD markers that could be scored with high confidence. Based on these markers, genetic similarity coefficients were calculated, and a dendrogram was constructed using the unweighted pair-group method with arithmatic average (UPGMA). The analysis delineated three major clusters. The first cluster consisted of a group of five watermelon cultivars, a group of C. lanatus var. lanatus accessions and a group of C. lanatus var. lanatus accessions that contained some C. lanatus var. citroides genes. The second cluster consisted of the C. lanatus var. citroides accessions, while the third cluster consisted of the C. colocynthis accessions. The two C. lanatus clusters differentiated from each other and from the C. colocynthis cluster at the level of 58.8% and 38.9% genetic similarity. Our results indicate that closely related Citrullus PIs may have resistances to the same diseases. Thus, molecular markers may be a useful tool in the development of core collections of Citrullus PIs with resistance to diseases.

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RNA isolation from ripe fruit can be complicated by high concentrations of sugar and water. These sugars interfere with RNA extraction often resulting in low RNA quality and quantities, and high water concentrations dilute the RNA, making isolation difficult. We report a simple but novel method by which the majority of the excess sugar and water in mature fruit of tomato (Lycopersicon esculentum Mill.), watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai], and muskmelon (Cucumis melo L.) can be easily removed from tissue before RNA extraction. This method produced quality RNA in a shorter time than the currently accepted method for fruit tissue RNA isolation and does not require liquid nitrogen or a freeze dryer.

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The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), often causes serious damage to watermelon (Citrullus spp.), and there is a need to evaluate and identify watermelon germplasm resistant to T. urticae. Watermelon cultivars (Citrullus lanatus var. lanatus), and U.S. plant introduction (PI) accessions of C. lanatus var. citroides and C. colocynthis, were evaluated for preference by T. urticae (number of adults and eggs on leaves). In open-choice experiments in the greenhouse and in laboratory rearing cages, there was a significant preference by T. urticae for watermelon cultivars, Citrullus lanatus var. lanatus PIs, and C. lanatus var. citroides PIs over C. colocynthis PIs. All watermelon cultivars and PIs were infested, but the C. colocynthis PIs were significantly less infested with T. urticae. The C. colocynthis PIs may be useful sources for enhancing T. urticae resistance in cultivated watermelon.

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Powdery mildew has been reported on Citrullus lanatus in Africa and Europe for the past nine years, and in the United States for the past 6 years. During this time, it has occurred in the main watermelon production areas in the U.S. and has been documented in nine states (South Carolina, Georgia, Florida, Oklahoma, Texas, Maryland, New York, Arizona, and California). This is of great concern to the watermelon industry since powdery mildew is difficult to control and can have a severe impact on yield and fruit quality due to loss of photosynthetic area and sunscald. Finding resistant C. lanatus germplasm is needed for the development of commercial varieties containing this resistance. This report summarized the status of an ongoing project to screen the entire USDA–ARS C. lanatus germplasm collection. Currently, the collection is being screened for race 1 and race 2 Podosphaera xanthii (syn. Sphaerotheca fuliginea auct. p.p.), the causal agent of powdery mildew in C. lanatus in the United States. Resistance genes appear to exist for both races and the genes conferring resistance to race 1 appear to be different than race 2 resistance genes. Allelism tests are currently in process to determine the number of resistance genes present.

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