Mark W. Farnham
Using anther culture to generate doubled-haploid (DH) homozygous lines for use as parents in F1 hybrid crosses has become a common practice in breeding broccoli (Brassica oleracea L. Italica Group). During anther culture and subsequent embryogenesis and plant regeneration, polyploidization of microspore-derived embryos may not occur or it may occur accompanied by a doubling, tripling, quadrupling, octupling, or irregular polyploidization of the genome. Thus regenerants from the process can be haploids, diploids, triploids, tetraploids, octaploids, or aneuploids. The objectives of this research were to 1) conduct repeat cycles of broccoli anther culture using a group of F1 hybrids as anther donors and develop populations of regenerants; 2) analyze resulting populations using DNA flow cytometry and determine the influence of F1 source on frequency of different ploidy levels among regenerants; and 3) compare seed set in broccoli inbreds developed in a traditional selfing program compared to seed set in DH broccoli derived from anther culture. In two cycles (1994 and 1995) of anther culture, anther-derived populations of regenerants were developed using the F1 hybrids `Marathon', `Everest', `High Sierra', and `Futura' as sources of anthers. In 1994, `Everest', `High Sierra', and `Futura' yielded populations that included 2% to 7% haploids, 53% to 56% diploids, 32% to 38% tetraploids, and 5% to 6% other types. `Marathon'-derived regenerants were 5% haploid, 78% diploid, 15% tetraploid, and 2% other, showing significantly more diploids. In 1995, `Marathon' regenerants again included significantly more diploids and fewer tetraploids than those derived from other F1 sources, confirming that the genotype of the anther source affects the frequency of a particular ploidy level among regenerants derived from culture. In manual self-pollinations of 1994 regenerants, only diploids and rare tetraploids set seed. When plants that set no seed were discounted, seed production following manual self pollinations of 1995 regenerants was not significantly different from that of traditional inbreds derived from the same F1 sources.
Mark W. Farnham
A collection of collard (Brassica oleracea L., Acephala group) germplasm, including 13 cultivars or breeding lines and 5 landraces, was evaluated using randomly amplified polymorphic DNA (RAPD) markers and compared to representatives of kale (Acephala group), cabbage (Capitata group), broccoli (Italica group), Brussels sprouts (Gemmifera group), and cauliflower (Botrytis group). Objectives were to assess genetic variation and relationships among collard and other crop entries, evaluate intrapopulation variation of open-pollinated (OP) collard lines, and determine the potential of collard landraces to provide new B. oleracea genes. Two hundred nine RAPD bands were scored from 18 oligonucleotide decamer primers when collard and other B. oleracea entries were compared. Of these, 147 (70%) were polymorphic and 29 were specific to collard. Similarity indices between collard entries were computed from RAPD data and these ranged from 0.75 to 0.99 with an average of 0.83. Collard entries were most closely related to cabbage (similarity index = 0.83) and Brussels sprouts entries (index = 0.80). Analysis of individuals of an OP cultivar and landrace indicated that intrapopulation genetic variance accounts for as much variation as that observed between populations. RAPD analysis identified collard landraces as unique genotypes and showed them to be sources of unique DNA markers. The systematic collection of collard landraces should enhance diversity of the B. oleracea germplasm pool and provide genes for future crop improvement.
Mark W. Farnham
Broccoli (Brassica oleracea L. Italica group) breeders are increasingly using anther or microspore culture to produce dihaploid (diploid), homozygous lines for use in making hybrids. During the process of anther culture and subsequent plant regeneration, wherein embryos develop from microspores and plants are regenerated from the embryos, 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, instead of a doubling. Thus, populations may contain haploids, triploids, or tetraploids, in addition to diploids. In two cycles (1994-95 and 1995-96) of anther culture, regenerated populations from different broccoli hybrid sources were evaluated using flow cytometry to facilitate efficient identification of diploids vs. haploids, tetraploids, or others and to determine if anther donor genotype has an effect on the frequency of different ploidy levels among regenerants. In the first cycle, five broccoli hybrids had anther-derived populations in which ≈33% were haploid, 55% diploid, 37% tetraploid, and 5% aneuploid or abherent types. The hybrid, `Marathon', was different; it's regenerants were 78% diploid and only 15% tetraploid. In the second cycle, anther-derived populations had a significantly different makeup with a most hybrids giving 30% to 40% diploids and 50% to 60% tetraploids. However, consistent with the previous cycle, `Marathon' gave significantly more diploids (68%) and fewer tetraploids (25%) than other hybrids. These results indicate that anther donor genotype affects ploidy frequency among regenerants. Genotypes producing a high frequency (>60%) of diploids may be relatively uncommon.
Mark W. Farnham
Collard (Brassica oleracea L. var. acephala) is an important vegetable the southeastern U. S. There are few (about 10) commercial cultivars, half being open-pollinating (OP) lines, the remainder more recent F1 hybrids. There is a potential untapped B. oleracea germplasm pool in the form of collard landraces perpetuated by southeastern gardeners and farmers. To determine the amount of genetic variation among cultivars and also whether landraces represent unique genotypes, ten cultivars and eight lines or landraces were evaluated using RAPD analysis. Decamer primers were used to amplify total genomic DNA and to differentiate collard lines and other B. oleracea crop cultivars. Additionally, individuals of an OP collard cultivar and a land-race were analyzed to evaluate intra-line variation. Virtually all primers detected polymorphic bands among lines although some identified considerably more variants. Intra-line analysis indicated that OP lines are genetically broad-based populations. Many unique RAPD markers were identified in landraces indicating that the lines represent unique genotypes and that further line collection is warranted.
Anna Hale and Mark W. Farnham
Private and public vegetable breeders are interested in using current and emerging PCR-based marker systems in their respective improvement programs. However, before new systems are employed to replace existing ones, the new systems must prove to be efficient and cost-effective alternatives. Sequence related amplified polymorphisms (SRAPs), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) were compared for their ability to differentiate individuals of a diverse group of 24 elite broccoli (Brassica oleracea L. italica) inbreds. Genomic DNA was assayed using 24 AFLP, 24 SRAP, and 44 SSR primer pairs. In this assessment, SSRs produced an average of only two bands per primer, with 25% of these bands being monomorphic, and the remaining bands detecting very few differences among the inbreds. Although the AFLP method resulted in a lower rate (63%) of polymorphism than the SSRs, it produced about 20 bands per primer. SRAPs produced an average of 14 bands per primer, with 82% of these bands being polymorphic. Since AFLP and SRAP markers had a higher multiplex ratio and SSRs were frequently monomorphic, AFLP and SRAPs were more effective in differentiating the elite broccoli inbreds examined in this study. Similarity matrices were generated from the AFLP and SRAP data, and resulting dendographs were compared.
Mark W. Farnham and Thomas Bjorkman
Breeding a vegetable crop for adaptation to a temperature regime that is higher than the recognized optimum for the species in question is an example of breeding for abiotic stress tolerance. Before embarking on a project to breed for such stress tolerance, we propose that several critical considerations or questions must be addressed. These considerations include the following: 1) What is the effect of the abiotic stress on the crop to be improved; 2) what will be the conditions of the selection environment; 3) what germplasm is available that contains the necessary genetic variation to initiate improvement; 4) what breeding scheme will be used to facilitate improvement; and 5) what will be the specific goals of the breeding effort? We use a case study with broccoli to breed for adaptation to high-temperature environments to provide examples of how each of these considerations might be addressed in developing an improvement effort. Based on documented success with this case study in which broccoli quality and performance under high-temperature summer environments has been improved, insights are provided that should be useful to future attempts to breed vegetables more tolerant of an abiotic stress.
Min Wang and Mark W. Farnham
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.