Downy mildew, caused by the fungal parasite Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of Brassica oleracea L. crops, including broccoli ( B. oleracea , Italica Group). The development and deployment of downy mildew resistant broccoli cultivars is a priority for breeders and producers. Identification of genetic markers linked to downy mildew resistance genes should facilitate selection for resistance and pyramiding of resistance genes into cultivars. The objectives of this study were to 1) identify RAPD markers linked to a single dominant gene for resistance in broccoli, 2) clone and sequence the linked RAPD markers, and 3) develop and evaluate SCAR markers as screening tools for resistance. Bulked segregant analysis led to the identification of eight linked RAPD markers following a screen of 848 decamers. Two of the linked RAPD fragments, UBC359 620 and OPM16 750 , were converted to dominant SCAR markers linked in coupling to the resistance locus at 6.7 and 3.3 cM, respectively. The SCAR marker based on UBC359 620 sequence exhibited less accuracy (94%) than the original RAPD (96%) in differentiating resistant and susceptible plants, but the accuracy (97%) of the OPM16 750 -SCAR was not different than the original RAPD. These SCAR markers are among the first genetic markers found linked to a gene conferring cotyledon-stage downy mildew resistance in B. oleracea . Results of this work provide breeders with useful information and tools for the systematic development of resistant cultivars.

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 F 1 hybrid. Subsequently, F 2 and backcross (BC) populations were developed from the hybrid. In addition, a DH population of ≈100 lines was developed from the same F 1 used to create the F 2 and BC. All populations were evaluated for response to artificial inoculation with P. parasitica at the three- to four-leaf stage. F 1 plants were resistant like the resistant parent and F 2 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 F 1 hybrids and used commercially to prevent downy mildew.

Broccoli ( Brassica oleracea L., Italica Group) is a good vegetable source of Ca and Mg, two critical minerals in human nutrition. Studies have shown that bioavailability of Ca from broccoli is comparable to that from milk. Thus, broccoli is an important alternative source of Ca in segments of the population that consume limited amounts of dairy products. Essentially nothing is known about the genetic influence on variation in Ca or Mg concentration of broccoli heads. Thus, the goal of this research was to examine variation in Ca, and also Mg concentrations, in a collection of USDA inbreds and commercial F 1 hybrids. In 1996 and 1997 field studies, significant differences among inbred entries and among hybrid entries were observed for Ca and Mg concentrations of broccoli heads. With hybrids and inbreds, mean head Ca concentrations were ≈3.0 mg·g -1 dry weight (DW), and entries with lowest and highest Ca concentrations differed >2-fold. Mean Mg concentrations of hybrid heads was 2.3 mg·g -1 DW (range 1.8 to 2.6) and 2.8 mg·g -1 DW (range 2.2 to 3.7) in 1996 and 1997, respectively. Inbred lines had mean head Mg concentrations of 2.0 and 2.6 mg·g -1 DW in the two respective years and ranges in concentration were similar as for hybrids. Analysis of variance indicated significant environment and entry by environment effects for Ca and Mg concentrations of hybrids. With inbreds, a significant entry by environment effect for Ca concentration and environment effect for Mg concentration was also observed. Significant environment and entry by environment effects indicate that the environmental influence on phenotypic expression of Ca and Mg concentrations may complicate genetic improvement of head mineral concentration.

Downy mildew, caused by Peronospora parasitica (Pers. ex Fr.), is one of the most economically important diseases in broccoli (Brassica oleracea L. Italica group). Previous studies reported that resistance to downy mildew in broccoli depends on plant age and that seedling resistance appears to be independent of mature-plant resistance. The objectives of our studies were to evaluate resistance and susceptibility of USDA broccoli inbreds to downy mildew and to investigate the interaction between the host and pathogen at two plant stages with single or double inoculation. Multiple screening tests at both cotyledon and three-expanded leaf stages using 38 entries, including USDA inbreds and commercial hybrids, were conducted in randomized complete-block designs. In these tests, every leaf of each plant was thoroughly sprayed with P. parasitica isolate PP1 at a concentration of 10,000 sporangia per ml at both stages. Ratings for downy mildew reaction phenotype were made at 9 days postinoculation on a 0-9 scale of increasing disease severity. We found significant phenotypic variation to infection among broccoli entries. We observed three general phenotypes: 1) resistance at both stages; 2) susceptible at cotyledon stage combined with resistance at three-expanded leaf stage; and 3) susceptibility at both stages. Additionally, inoculation at the cotyledon stage had no effect on inoculation at the three-expanded leaf stage.

Anther and microspore culture are commonly utilized to produce doubled-haploid (diploid), homozygous lines in broccoli (Brassica oleracea L. Italica Group). It is well-documented that doubled-haploid regenerants are produced by means of polyploidization during anther culture. However, polyploidization may not occur at all, or it may involve a tripling or quadrupling of the chromosome complement. As a consequence, regenerated populations from anther culture contain diploids, but also haploids, triploids, and tetraploids. Microspore culture represents a simpler and more direct method for producing doubled-haploids. Although a similar mix of ploidy types is likely to be observed among regenerants derived from microspore culture, the actual ploidy levels of such regenerants have not been documented for broccoli. Thus, the objectives of this study were to compare ploidy levels of regenerants developed using both anther and microspore culture in broccoli, and to examine phenotypic variation in ploidy makeup of populations developed from both anther and microspore culture using different F1 hybrids. Broccoli regenerants were derived simultaneously from both anther and microspore cultures using the same four F1 hybrids, including Everest, Patriot, Greenbelt and Major. Ploidy level was determined by flow cytometry. A majority of regenerants derived from both anther and microspore culture, were determined to be diploids or tetraploids. Significant differences in ploidy makeup of populations were observed among hybrid varieties for both culture techniques. Regardless of the culture method used, `Everest' produced a greater percentage of diploids and a lower percentage of tetraploids than `Patriot' did. Haploids were observed more frequently from microspore culture than from anther culture when `Everest' and `Major' served as parents.