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Kanta Kobira, Khalid Ibrahim, Elizabeth Jefferey, and John Juvik

Considerable epidemiological evidence exists on the association between consumption of antioxidant-rich vegetables and incidence of chronic diseases, including cancer and cardiovascular disease. Broccoli (Brassica oleracea L. sp. italica) florets are relatively abundant sources of antioxidants, and potentially amenable to genetic manipulation to enhance this vegetable's health-promoting properties. This investigation focuses on the identification of chromosomal segments in the nuclear genome of broccoli associated with antioxidant carotenoid and tocopherol variability. A broccoli F2:3 population consisting of 163 families derived from a cross between two parents (VI-158 and BNC) and previously mapped with 62 polymorphic SSR and SRAP marker loci was evaluated for carotenoid and tocopherol concentration in floret tissue over two growing seasons. Significant differences were observed among F2:3 family means for concentrations of lutein (10-fold difference between the lowest and highest family), beta-carotene 17-fold), alpha-tocopherol (8-fold) and gamma-tocopherol (6-fold). On a concentration basis, beta-carotene, lutein, alpha-tocopherol, and gamma-tocopherol were the most abundant antioxidant forms in broccoli. Heritability estimates of primary phytochemicals ranged from 0.35 to 0.38, 0.40, and 0.44 for beta-carotene, alpha-tocopherol, gamma-tocopherol, and lutein, respectively. Composite interval mapping (CIM) identified two quantitative trait loci (QTL) associated with carotenoid variability on two linkage groups and five QTL associated with tocopherol variability on four linkage groups. The QTL identified in this study have potential for use in marker-assisted crop improvement programs to develop elite germplasm designed to promote health among the consuming public.

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Antonio Figueira, Jules Janick, and Peter Goldsbrough

RAPD markers were used to examine genetic similarity in cacao. DNA from 30 cacao cultivars amplified using 15 arbitrary oligonucleotide primers, produced a total of 112 fragments, of which 88% were polymorphic. A phenogram was developed which illustrates the genetic relationships among the cacao cultivars representing the four major geographic groups of cacao (Criollo, Trinitario, Forastero Lower Amazonian, and Forastero Upper Amazonian). The phenogram indicated a general separation of the four groups into three clusters. Criollos and Trinitarios (supposedly hybrids between Forastero and Criollos types) appeared in a single cluster. Lower Amazonian cultivars (mainly selections made in Bahia, Brazil) appeared in a separate cluster. The third cluster consisted of the Upper Amazonian cultivars, which were originally collected from the region believed to be the center of origin of this crop. This cluster displayed the furthest genetic distance from the others. Crosses between Upper Amazon germplasm and local selections have shown heterosis in clonal crosses, which has been exploited in all genetic improvement programs for cacao. We propose that genetic distances based on RAPD markers can be potentially used as a criterion to select parents capable of producing superior hybrids and populations. Genetic relationships can also be useful to define germplasm collections and conservation strategies. Studies are underway to compare phenograms derived from RAPD markers and ribosomal RNA gene polymorphisms.

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Mirko Siragusa, Fabio De Pasquale, Loredana Abbate, and Nicasio Tusa

A collection of 18 accessions of sour orange (Citrus aurantium L.) coming from Sicily and other countries was investigated by two polymerase chain reaction (PCR)-based DNA marker technologies. Ten inter-simple sequence repeat (ISSR) primers and fifteen randomly amplified polymorphic DNA (RAPD) primers were used to identify and to evaluate the genetic variability and relationship of accessions. A total of 111 ISSR and 145 RAPD amplified fragments were used to estimate the Dice's coefficient of similarity for cluster analysis using a unweighted pair-group method using an arithmetic averaging (UPGMA) algorithm. The genetic relationships identified using ISSR and RAPD markers were highly concordant, such that the correlation between ISSR and RAPD genetic distance (GD) estimates was r = 0.93. The ISSR and RAPD analysis of 18 sour orange accessions found a high grade of genetic diversity in foreign accessions, while a low variability was detected in local accessions. Sicilian accessions could be grouped in two distinct clusters, including indistinctly plants from three origin regions. Some markers could be linked to the different growing areas. The ISSR and RAPD molecular reference system seems to be suitable for a fine identification of tightly related plants and the obtained results can form the basis for future setting up of Citrus rootstock genetic improvement projects.

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Jan G. Tivang, Neal DeVos, James Nienhuis, and Paul Skroch

Individual heads (capitula) from five discrete artichoke, Cyara scolymus L., populations were evaluated using RAPD markers. One vegetatively-propagated cultivar; Green Globe; two seed-propagated cultivars, Imperial Star and Big Heart XR-1; and two breeding populations were examined. Twenty-seven RAPD primers were scored yielding 2 to 16 polymorphic bands resulting in a total of 178 bands. Our objective was to determine if RAPD markers could be used to distinguish between and within populations. The genetic relationships among populations as well as among individuals within each population were estimated using the ratio of discordant to total bands scored. Data reduction (MDS) provided a plot indicating five clusters corresponding to the five populations. Confirmation of the presence of five discrete clusters was obtained by analysis of variance of the marker frequencies. The genetic diversity index (GDI) was calculated for each populations as the pooled variance of band frequency for each population. The GDI values were highly correlated to the mean genetic distance within each population. The homogeneity of variance for the GDI values associated with each population were compared using the Siegel-Tukey test for homogeneity of spread.

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Frank Cheng, Norman Weeden, and Susan Brown

The ability to pre-screen apple populations for fruit color at an early seedling stage would be advantageous. In progeny of the cross `Rome Beauty' × `White Angel' red/yellow color variation was found to be highly correlated with the genotype at Idh-2, an isozyme locus that was heterozygous in both parents. We postulate that the red/yellow color variation was produced by a single gene linked to I&-2 and also heterozygous in both parents. This population was also screened with over 400 primers to detect randomly amplified polymorphic (RAPD) markers for fruit color. DNA extraction procedures were developed for bark, and DNA was extracted from bark samples and leaves. Red and yellow fruited individuals were examined in bulk. Several markers have been found that are linked to red color. A high density map is being constructed in this region. These markers are being examined in other crosses segregating for fruit color. The application of these markers will be discussed in relation to the inheritance and manipulation of fruit color.

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David Shupert, Aaron P. Smith, Jules Janick, Peter B. Goldsbrough, and Peter M. Hirst

The codominant PCR marker AL07-SCAR closely linked to the Vf gene for scab resistance was used to genotype seedlings in three apple populations in which each parent (`GoldRush', `Enterprise', `Pristine', and CQR10T17) was resistant to apple scab. The marker was used to predict the genotype at the Vf locus. Each parent was heterozygous. In two populations (CQR10T17 × `GoldRush' and `Pristine' × `GoldRush') seedlings segregated 1:2:1 for fragments associated with VfVf:Vfvf:vfvf as predicted by Mendelian segregation. However, in another population (`GoldRush' × `Enterprise') the ratio was 1.5:1:1.5, suggesting some type of selection against heterozygotes. Fruiting seedlings were rated for the presence of fruit scab. No scab was observed on seedlings homozygous for the PCR marker linked to Vf, a small amount of scab was observed on one heterozygous seedling out of 35, and 22 of 26 seedlings that were homozygous recessive, had fruit scab.

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Margaret R. Pooler, Louise G.H. Riedel, S.E. Bentz, and A.M. Townsend

Controlled pollinations were made between five hemlock (Tsuga) species from eastern North America and Asia, resulting in over 5700 germinating seedlings. A subset of putative hybrid seedlings from each cross was tested for authenticity by various DNA marker systems. The most reliable and useful system for verifying hybrids was amplified fragment-length polymorphism (AFLP) markers. Hybridizations between the eastern North American species, T. canadensis [L.] Carriere and T. caroliniana Engelm., and the Asian species, T. chinensis (Franch.) E. Pritz., were used as a model to test the inheritance, reliability, and ease of use of these markers. Using AFLP markers, we were able to verify 58 hybrids between T. caroliniana and T. chinensis, one hybrid between T. caroliniana and T. canadensis, but could find no definitive hybrids between T. canadensis and T. chinensis. Results using other marker systems, including RAPD, SCAR, ITS, and SSR, are also presented.

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Soon O. Park, Dermot P. Coyne, and James R. Steadman

Bean rust, caused by Uromyces appendiculatus, is an important disease of common bean (Phaseolus vulgaris L.). The objective was to identify RAPD markers linked to the gene (Ur-6) for specific resistance to rust race 51 using bulked segregant analysis in an F2 segregating population from the common bean cross pinto `Olathe' (resistant to rust) × great northern Nebraska #1 selection 27 (susceptible to rust). A single dominant gene controlling specific resistance to race 51 was hypothesized based on F2 segregation, and then was confirmed in the F3 generation. A good fit to a 3:1 ratio for band presence to band absence for each of three markers was observed in 100 F2 plants. Three RAPD markers were detected in a coupling phase linkage with the Ur-6 gene. Coupling-phase RAPD marker OAB14.600 was the most closely linked to the Ur-6 gene at a distance of 3.5 cM among these markers. No RAPD markers were identified in a repulsion phase linkage with the Ur-6 gene. The RAPD markers linked to the gene for specific rust resistance of Middle American origin detected here, along with other independent rust resistance genes from other germplasm, could be utilized to pyramid multiple genes into a bean cultivar for more durable rust resistance.

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R.N. Trigiano, K.M. Kaveriappa, S.E. Schlarbaum, M.T. Windham, and W. Witte

DNA amplification fingerprinting (DAF) was Used to characterize both parents (different cultivars) in breeding experiments with Cornus florida. Putative hybrids were fingerprinted and true crosses identified by finding unique male parent products in amplification profiles. Both manual and honey bee mediated pollinations successfully produced hybrid seed. Axillary buds from seedlings were used to initiate proliferating shoot cultures on woody plant medium with 4.5 μm BA. Initiation and development of adventitious roots were dependent on IBA (4.1 μm), sucrose (0–2%), and agar (0.2–0.6%) concentrations. About 40–50% of the microshoots produced roots and were acclimatized to greenhouse conditions. Cultures have been maintained without loss of regeneration potential for over 2 years. Clonal material can be reentered into the breeding program or used to evaluate horticultural characteristics in different environments and locales.

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Tim Rinehart and Sandy Reed

Hydrangea popularity and use in the landscape has expanded rapidly in recent years with the addition of remontant varieties. Most cultivars in production belong to the species Hydrangea macrophylla but H. paniculata, H. arborescens, H. serrata, H. aspera, H. heteromalla, H. integrifolia, H. anomala, H. seemanii, and H. quercifolia are also commercially available. In addition to species diversity there is high intra-species variation, particularly in H. macrophylla, which includes mopheads, lacecaps, French, Japanese, dwarf, and variegated varieties. Relatively little is known about the genetic background or combinability of these plants. DNA sequence data, genome size, RAPD, AFLP, and ISSR markers have been used for taxonomic identification and to estimate diversity within the genus. All of these methods have limited usefulness in a large scale breeding program. We recently established microsatellite markers for Hydrangea and evaluated their utility for estimating species diversity and identifying cultivars within H. macrophylla and H. paniculata. We also verified an inter-specific cross between H. macrophylla and H. paniculata using these markers. Future research includes marker assisted breeding, particularly with respect to remontant flowering traits.