The Y2 locus conditions α- and β-carotene accumulation in the xylem (core) of carrot roots. The dominant allele suppresses carotene, but not xanthophyll accumulation, resulting in yellow-cored roots. Individuals homozygous for the recessive allele are rich in carotenes and are therefore orange-cored. Increased consumer interest in high carotene produce requires improved understanding of carotene biosynthesis and color development and more-efficient breeding techniques. We examined 103 F2 individuals generated from inbred populations differing in core carotene content. Bulked segregant analysis identified AFLP bands putatively linked to Y2. Linkage was confirmed for some bands by mapping. Linked bands were excised from gels, re-amplified, cloned into pGEM vectors, and sequenced. Cloned fragments and sequence information were used to characterize larger genomic regions to identify codominant markers. Currently we are developing codominant, PCR-based markers that can be used to rapidly genotype individuals in breeding programs, to characterize wild, feral, and cultivated populations for diversity and evolution studies, and to examine the role of Y2 in carotene accumulation.
James M. Bradeen and Philipp Simon
James M. Bradeen and Michael J. Havey
The genus Allium contains about 500 species, several of which have been cultivated for millennia. Despite its long history of cultivation and its worldwide economic importance, little is known phylogenetically about Allium. Identification of the likely progenitor of A. cepa (the bulb onion) will focus future collection efforts on wild germplasm that may be useful in the genetic improvement of the bulb onion. Several classification schemes based on morphological characteristics have been proposed for A. cepa and its presumed closest relatives. None of these schemes has been definitive. Nuclear restriction fragment length polymorphisms (RFLPs) were identified among Allium species in sections Cepa and Phyllodolon. These were used to unbiasedly estimate phylogenetic relationships.
James M. Bradeen and Michael J. Havey
Commercial bulb-onion (Allium cepa L.) growers often complain that hybrids they have grown successfully for a few years fail to perform at the expected level. Inbreds used to produce hybrid-onion seed rarely have been self-pollinated for more than two generations and retain a high level of heterozygosity. Over time, selection, drift, or contamination of inbreds may contribute to disappointing hybrid performance. We identified randomly amplified polymorphic DNA (RAPD) between two inbred onion lines, demonstrated their Mendelian inheritance, and tried to distinguish among and examine changes in independently maintained, publicly released inbred lines of onion. We observed poor agreement between data sets based on genetically characterized and uncharacterized RAPD markers. Our analyses used only genetically characterized RAPD markers and revealed that contamination, in addition to-drift and/or selection, likely contributed to differences among independently maintained, publicly released inbreds. However, RAPD markers were not able to distinguish confidently among four related inbreds. RAPD markers will be useful in Allium genetics and breeding, but identifying and characterizing reliable polymorphisms is critical.
Steven J. McKay, James M. Bradeen, and James J. Luby
The commercially successful apple (Malus pumila Mill.) cultivar Honeycrisp is known for its high degrees of crispness and juiciness. This cultivar has been incorporated into numerous breeding programs in an effort to duplicate its desirable texture traits in conjunction with such other traits as reduced postharvest disorders, disease resistance, and improved tree vigor. This study characterizes variability and estimates heritability for several apple fruit texture traits within a large breeding population over several years. Five full-sib families, all sharing ‘Honeycrisp’ as a common parent, were assayed with respect to crispness, firmness, and juiciness using sensory evaluation panels and total work required to fracture tissue using instrumental methods. The incomplete block design of the sensory panels, coupled with best linear unbiased prediction, facilitated the evaluation of a large number of genotypes with small numbers of fruit per genotype while accounting for individual sensory panelist effects. Broad-sense heritability estimates exceeded 0.70 for all four traits. Principal component analysis, applied to the phenotypic data, characterized ‘Honeycrisp’ as having average crispness and low firmness (53rd percentile relative to its offspring) but also as being a relatively extreme example of high juiciness and low work to fracture (first percentile). The improved characterization of desired fruit texture phenotypes and the high levels of broad-sense heritability provide valuable tools for the further development of new, high-quality apple cultivars.
Vance M. Whitaker, Stan C. Hokanson, and James Bradeen
Black spot, incited by the fungus Diplocarpon rosae Wolf, is the most significant disease problem of landscape roses (Rosa hybrida L.) worldwide. The documented presence of pathogenic races necessitates that rose breeders screen germplasm with isolates that represent the range of D. rosae diversity for their target region. The objectives of this study were to characterize the genetic diversity of single-spore isolates from eastern North America and to examine their distribution according to geographic origin, host of origin, and race. Fifty isolates of D. rosae were collected from roses representing multiple horticultural classes in disparate locations across eastern North America and analyzed by amplified fragment length polymorphism. Considerable marker diversity among isolates was discovered, although phenetic and cladistic analyses revealed no significant clustering according to host of origin or race. Some clustering within collection locations suggested short-distance dispersal through asexual conidia. Lack of clustering resulting from geographic origin was consistent with movement of D. rosae on vegetatively propagated roses. Results suggest that field screening for black spot resistance in multiple locations may not be necessary; however, controlled inoculations with single-spore isolates representing known races is desirable as a result of the inherent limitations of field screening.
Joseph J. King, James M. Bradeen, and Michael J. Havey
Nuclear RFLPs were used to estimate relationships among 14 elite commercial inbreds of bulb onion (Allium cepa) from Holland, Japan, and the United States. Variability for known alleles at 75 RFLP loci and 194 polymorphic fragments revealed by 69 anonymous cDNA probes and a clone of alliinase were scored to yield genetically characterized and uncharacterized data sets, respectively. The inbred onion populations possessed more than two alleles at 20 of 43 (46%) codominant RFLP loci. Relationships among the inbreds were estimated by cluster analysis of simple-matching (genetically characterized data) and Jaccard (genetically uncharacterized data) coefficients using the unweighted pair group method and agreed with known pedigrees. RFLPs confidently distinguished among elite inbreds within and between specific market classes. RFLP profiles for virtual hybrids were computer-generated by combining gametic arrays among inbreds of the same market class and analyzed as described above. Allelic and genetically uncharacterized RFLPs confidently distinguished among these hybrids, even though heterozygosity for many markers produced a majority of monomorphic fragments. We randomly sampled decreasing numbers of RFLPs from the complete data sets and calculated simple-matching and Jaccard distances, noting the numbers of probes that were unable to distinguish any two inbreds or hybrids. As few as 10 polymorphic probe-enzyme combinations distinguished among all the inbreds and samples of 20 genetically characterized or 10 genetically uncharacterized clones distinguished all the virtual hybrids. This study demonstrated that the previously reported few RFLPs observed among open-pollinated (OP) onion populations were due to the highly heterozygous nature of the OP population.
Michael J. Havey, Joseph J. King, James M. Bradeen, and Ockyung Bark
James M. Bradeen, S. Kristine Ness, Geraldine T. Haberlach, Susan M. Wielgus, and John P. Helgeson
Late blight of potato, caused by the fungal pathogen Phytophthora infestans, is of great economic significance and no important U.S. potato cultivars are reliably resistant. The diploid species Solanum bulbocastanum is highly resistant to late blight, even under extreme conditions, but is sexually incompatible with potato. We have generated potato + S. bulbocastanum somatic hybrids. These hexaploid hybrids are highly resistant to late blight and progeny from two successive backcrosses to cultivated potato are either fully resistant or susceptible. The advanced generations are morphologically similar to potato. We have initiated mapping efforts to identify bulbocastanum chromosomal regions responsible for late blight resistance. Using RFLPs, RAPDs, and AFLPs, we identified a single chromosomal region (i.e., single gene or group of linked genes) on chromosome 8 that accounts for 62.2% of the observed resistance. The tomato cDNA clone CT88 cosegregates with late blight resistance in our material and was used to isolate a homeologous BAC clone from a S. bulbocastanum library. Our current efforts include mapping resistance in both fusion-derived and diploid S. bulbocastanum materials via map merging. As markers linked to late blight resistance are identified, we will attempt to convert them to marker forms useful for large-scale breeding efforts. To date, we have successfully converted RAPD and RFLP markers to SCAR and CAPS marker forms. Finally, continued fine mapping and BAC clone characterization will enable future map-based cloning efforts.
Alexander Q. Susko, Timothy A. Rinehart, James M. Bradeen, and Stan C. Hokanson
Deciduous azaleas are an important element of residential and commercial landscapes in the United States after substantial trait improvements to increase their market appeal. Despite progress in breeding for ornamental characteristics and cold hardiness, intolerance to elevated pH and calcareous soils continues to limit their use in managed landscapes. Therefore, we assessed the utility of in vitro and greenhouse phenotyping approaches to evaluate and select for improved soil pH tolerance to increase the efficiency of breeding for this important trait. The research presented offers an example for implementing image-based phenotyping to expedite cultivar development in woody ornamental crops.
James M. Bradeen, Inga C. Bach, Mathilde Briard, Valérie le Clerc, Dariusz Grzebelus, Douglas A. Senalik, and Philipp W. Simon
A sample of 124 Daucus carota L. accessions, including cultivated carrot [D. carota ssp. sativus (Hoffm.) Arcangeli] and related wild subspecies, using a variety of molecular markers was examined. Represented within the samples were wild accessions from 18 countries, 14 of 16 major root types of European origin, and examples of major North American and Asian cultivated carrot types. Amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) markers revealed extensive variation within D. carota. Although cultivated carrot and wild D. carota subspecies can cross freely, cultivated and wild carrots clustered separately, supporting the possibility that human selection for desirable horticultural traits has artificially reduced gene flow between cultivated and wild forms. Our analyses support the likelihood that North American D. carota populations arose due to introduction of weedy materials rather than escape of cultivated forms. With the exception of wild vs. cultivated types, no genetic alliances were evident in dendrogram topology. Furthermore, between and even within nonmapped marker classes, dendrogram topology predictions were not consistent. Generally poor correlations among root types, geographic origin, mitochondrial, plastid, and specific nuclear diversity and AFLP/ISSR data were also observed. We concluded that genetic diversity in carrot is extensive and relatively nonstructured in nature.