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Pan-chi Liou, Fred G. Gmitter Jr., and Gloria A. Moore

Citrus genetic studies and cultivar improvement have been difficult with conventional techniques. Alternative approaches are needed to enhance efficiency of such studies. Our objectives were to characterize the Citrus genome and to initiate development of a linkage map using RFLP and isozyme analysis. Methods of Citrus DNA extraction were developed to allow the isolation of chromosomal DNA of acceptable quality for recombinant' DNA manipulations. A PstI Citrus genomic library was constructed to create DNA clones for the RFLP survey. A rapid, reliable procedure was developed to facilitate screening of the library for useful clones. The methods used and strategy followed minimized contamination with organelle DNA, increased the frequency of single copy clones, and allowed rapid screening of the newly–constructed library. Linkage relationships of 49. markers, including 36 RFLP and 6 isozyme loci, were analyzed and a map comprised of 8 linkage groups was constructed. Insertions or deletions were responsible for at least 30% of the RFLPs identified. A hypothesis of transposon activity in Citrus was proposed based on our observations.

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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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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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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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W.V. Baird, R.E. Ballard, S. Rajapakse, and A.G. Abbott

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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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Zhanao Deng, Jinguo Hu, Fahrettin Goktepe, Brady A. Vick, and Brent K. Harbaugh

Cultivated caladiums are valued for their bright colorful leaves and are widely used in containers and landscapes. More than 1500 named cultivars have been introduced during the past 150 years, yet currently only about 100 cultivars are in commercial propagation in Florida. Caladium tubers produced in Florida account for 95% of the world supplies. Loss of caladium germplasm or genetic diversity has been a concern to future improvement of this plant. In addition, the relationship among the available cultivars, particularly those of close resemblance, has been lacking. This study was conducted to assess the genetic variability and relationship in commercial cultivars and species accessions. Fifty-seven major cultivars and 15 caladium species accessions were analyzed using the target region amplification polymorphism marker technique. This marker system does not involve DNA restriction or adaptor linking, but shares the same high throughput and reliability with the amplified fragment length polymorphism system (AFLP). Eight primer combinations amplified 379 scorable DNA fragments among the caladium samples. A high level of polymorphism was detected among the species accessions as well as among cultivars. These markers allowed differentiation of all the cultivars tested, including those hardly distinguishable morphologically. Clustering analysis based on these DNA fingerprints separated the cultivars into five clusters and Caladium lindenii far from other caladium species. The availability of this information will be very valuable for identifying and maintaining the core germplasm resources and will aid in selecting breeding parents for further improvement.

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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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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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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.