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Amnon Levi and Claude E. Thomas

A genetic linkage map was previously constructed for watermelon using a wide testcross population [{Plant Accession Griffin 14113; Citrullus lanatus var. citroides (L.H. Baiely) Mansf.} × the watermelon cultivar New Hampshire Midget; NHM {(Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus)} × United States Plant Introduction (PI) 386015 {Citrullus colocynthis (L.) Schrad.}]. One-hundred forty-six markers [randomly amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and sequence-related amplified polymorphism (SRAP) markers] unique to NHM and representing different linkage groups on the map were tested for polymorphism among 24 watermelon cultivars limited in genetic diversity. Five (9.4%) of 53 RAPD, six (40.0%) of 15 ISSR, 30 (81.0%) of 37 AFLP, and 33 (80.5%) of 41 SRAP markers tested produced polymorphism among the 24 cultivars. The polymorphic markers used in this study are scattered throughout the watermelon genome. However, a large number (19 of the 30) of AFLP markers clustered on one linkage group on the map. The SRAP markers proved to be most effective in producing polymorphism and in representing different linkage regions of watermelon genome. The polymorphic markers represent all 10 large linkage groups and five of the nine small linkage groups (altogether 15 of 19 linkage groups) of the genetic linkage map constructed so far for watermelon. These polymorphic markers can be useful in DNA fingerprinting of cultivars, in testing seed purity of breeding lines, and in identifying triploid (seedless) hybrid watermelons derived from crosses between closely related tetraploid and diploid lines.

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Claude E. Thomas and E.L. Jourdain

Evaluations for resistance against race 2 of downy mildew, incited by Peronospora parasitica, were conducted on 240 U.S. Plant Introductions (PI) classified as Brassica oleracea var. botrytis (consists of both broccoli and cauliflower types). Plants were inoculated at the two-expanded leaf stage with 5.0 × 103 sporangia per ml. Inoculated plants were incubated in a dark 16C dew chamber for 24 hr and were then placed in a 22C growth chamber with a 12-hr photoperiod. On the 7th day after inoculation, plants were returned to the dew chamber for 30 hr and ratings for downy mildew reaction phenotypes were made at 9 days postinoculation on a 0-9 scale of increasing disease severity. A disease index (DI) was calculated for each entry. Based on the DI, no PI entries were highly resistant. PI entries 181860, 188562, 204765, 204768,204772,204773, 204775,204779,241612, 264656,291567,373906, and 462225 were moderately resistant. (DI of 3.1-5).

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Claude E. Thomas and E.L. Jourdain

Field evaluations for resistance against downy mildew, incited by Pseudoperonospora cubensis [(Berk. and Cart.) Rostow], were conducted on 942 U.S. Plant Introductions (PI) of Cucumis melo L. (melon). A disease index (DI) was calculated for each entry. Based on DI, PI 124112 was highly resistant (DI = 3.7), and PIs 124111, 122847, 124210, 145594, and 165525 were resistant (DI = 3.0, 2.8, 2.6, 2.7, and 2.5, respectively). PIs 124111 and 124112 had one or more plants that exhibited a highly resistant reaction type (RT 4). Resistant (RT 3) plants were identified in 31 accessions, and 49 accessions bad moderately resistant (RT 2) plants.

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Claude E. Thomas and E.L. Jourdain

Evaluations for resistance against race 2 of downy mildew, incited by Peronosporu parasitica Pers. ex Fr., were conducted on 325 U.S. Plant Introductions (PI) classified as Brassicu oleracea var. capitata L. (cabbage). A disease index (DI) was calculated for each entry. In 77 of the tested PIs, from 2% to 100% of the plants had reaction phenotypes ≥ 3. The DIs for 24 PIs were significantly lower than the DI for the most resistant cabbage control, `Headstart' (DI = 5.9). Eight of these PIs were highly resistant, because all plants had a reaction phenotype ≥ 3. However, most of these eight are apparently misclassified as B. oleracea var. capitata.

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Claude E. Thomas, Amnon Levi, and Ellis Caniglia

Two hundred sixty-six Citrullus lanatus (Thumb.) Matsum. & Nakai accessions (Plant Introductions and named cultivars) were tested against a race 2 Sphaerotheca fuliginea (Schlechtend.: Fr.) Pollacci isolate to evaluate for resistance to powdery mildew disease. Growth room-grown seedlings were artificially inoculated with conidia from watermelon host leaves at 2-day intervals from the appearance of the first true leaf until test results data were taken, when the second true leaf was fully expanded. Plants were evaluated on a 1 to 9 scale of increasing disease severity. Disease indices (DIs) were calculated as weighted averages for each entry. All genotypes with resistant plants (powdery mildew rating 1 to 3) were reevaluated in a replicated test of 3 replications of 10 plants each. Disease indices were again calculated. Twenty-two plant introductions (PIs) and one named variety displayed intermediate resistance to powdery mildew in the replicated test with DIs ranging from 5.0 to 6.0.

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W. Patrick Wechter, Ralph A. Dean, and Claude E. Thomas

Two 24-mer primers, MUSKFOM I and MUSKFOM II, were developed that amplify a 1.5-kb DNA fragment in race 1 Fusarium wilt resistant muskmelon (Cucumis melo L.), but not in race 1 susceptible germplasm tested. Three race 1 resistant cultivars and two race 1 resistant breeding lines as well as eight race 1 susceptible lines were analyzed using the two sequence-specific primers in the polymerase chain reaction. These primers should prove valuable for nondestructive determination of Fom 2 gene introgression in breeding programs.

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Amnon Levi, Claude E. Thomas, Todd C. Wehner, and Xingping Zhang

Genetic diversity and relatedness were assessed among 46 American cultivars of watermelon (Citrullus lanatus var. lanatus), and 12 U.S. Plant Introduction accessions (PIs) of Citrullus sp. using 25 randomly amplified polymorphic DNA (RAPD) primers. These primers produced 288 distinct reproducible bands that could be scored with high confidence among cultivars and PIs. Based on the RAPD data, genetic similarity coefficients were calculated and a dendrogram was constructed using the unweighted pair-group method with arithmetic average (UPGMA). The cultivars and C. lanatus var. lanatus PIs differentiated at the level of 92% to 99.6% and 88% to 95% genetic similarity, respectively. In contrast, the C. lanatus var. citroides, and C. colocynthis PIs were more divergent and differentiated at the level of 65% to 82.5% and 70.5% genetic similarity, respectively. The low genetic diversity among watermelon cultivars in this study emphasizes the need to expand the genetic base of cultivated watermelon.

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Mark W. Farnham, Ellis J. Caniglia, and Claude E. Thomas

Broccoli (Brassica oleracea L. Italica group) breeders routinely use anther or microspore culture to produce dihaploid (diploid), homozygous lines. During the culture process, polyploidization occurs and diploid regenerants can result. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement. Thus, regenerated populations must be screened to identify the diploids that are the regenerants most likely to set seed and serve as inbred lines. DNA flow cytometry has proven a useful procedure for determining ploidy of anther derived regenerants. This study was undertaken to evaluate the effect of leaf age and sampling procedures on ploidy determination via flow cytometry. Anther-derived plants were analyzed at a four- to five-leaf stage (transplant stage) and at time of heading (mature plant stage). In addition, leaves were sampled on a given date and stability of the flow cytometry preparations was evaluated over 7 days. Lastly, the stability of ploidy readings of leaves stored at 4°C was examined over a 7-day period. In only one case out of 123 comparative assays did leaf age affect ploidy determination. For that exception, a haploid at transplant stage was a diploid at the mature plant stage. Flow cytometry preparations and also leaves stored at 4°C gave consistent ploidy determinations up to four days after preparations were made or tissue was refrigerated, respectively. These results indicate that broccoli breeders can make flow cytometry preparations on site and send them offsite for flow cytometry analysis. Alternatively, leaves could be refrigerated, sent offsite, and then prepared and analyzed at another location.

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Min Wang, Mark W. Farnham, and Claude E. Thomas

Downy mildew, caused by Peronospora parasitica (Pers. Fr.) Fr., is one of the most economically important diseases of broccoli (Brassica oleracea L. Italica group). Previous studies have shown that resistance to downy mildew in broccoli is dependent on plant age with seedling resistance being independent of mature-plant resistance. The objectives of this study were to: 1) determine if valid evaluations for downy mildew resistance can be conducted at both the cotyledon and the three to four true-leaf stages on the same plants of a given broccoli entry; 2) determine if doubled-haploid (DH) lines derived from the resistant hybrid `Everest' also exhibit resistance to downy mildew and if so, characterize the resistance phenotype(s) in these lines; and 3) determine if identified resistant DH lines exhibit resistance to isolates of P. parasitica acquired from different geographic regions of the United States. Twenty-three DH broccoli inbreds and two commercial hybrids were evaluated for reaction at different developmental stages to infection by P. parasitica in a controlled environment. Results showed that broccoli plants can be evaluated for downy mildew resistance in a two-stage process. Inoculation at the cotyledon stage did not offer any cross-protection or otherwise influence the expression of reaction phenotype (RP) when the same plants were subsequently inoculated at the three to four true-leaf stage. Three different RPs to infection by P. parasitica were identified in DH inbreds. These were: 1) susceptibility at both the cotyledon stage and the true-leaf stage; 2) resistance at both the cotyledon and true-leaf stage; and 3) susceptibility at the cotyledon stage but resistance at the true-leaf stage. There was no effect of two pathogenic isolates from different geographic regions on RP of DH broccoli inbreds. Selection of plant resistance to downy mildew at the cotyledon stage will effectively identify plants with high levels of resistance at subsequent developmental stages.

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Min Wang, Mark W. Farnham, and Claude E. Thomas

Downy mildew, incited by the biotrophic fungal parasite, Peronospora parasitica (Pers. Fr.) Fr., is one of the most destructive diseases of broccoli (Brassica oleracea L., Italica Group) and other related crop species throughout the world. Cultivation of resistant cultivars is the most desirable control method because it provides a practical, long-term, and environmentally benign means of limiting damage from this disease. The commercial hybrid cultivar, Everest, has been shown previously to contain a high level of downy mildew resistance. Doubled-haploid (DH) lines developed from that hybrid were also shown to exhibit a similar, high level of resistance at the three- to four-leaf stage. To determine the mode of inheritance of this true leaf resistance, the resistant DH line was crossed to a susceptible line (derived from `Marathon') to produce an F1 hybrid. Subsequently, F2 and backcross (BC) populations were developed from the hybrid. In addition, a DH population of ≈100 lines was developed from the same F1 used to create the F2 and BC. All populations were evaluated for response to artificial inoculation with P. parasitica at the three- to four-leaf stage. F1 plants were resistant like the resistant parent and F2 populations segregated approximately nine resistant to seven susceptible. Using the resistant parent as recurrent parent, BC populations contained all resistant plants, while the BC to the susceptible parent fit a 1 resistant: 3 susceptible segregation ratio. These results can be explained by a model with two complementary dominant genes. This model was confirmed by the DH population that segregated ≈1:3, resistant to susceptible. Due to the dominant nature of this resistance, controlling genes should be easily incorporated into F1 hybrids and used commercially to prevent downy mildew.