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Robert R. Krueger and Mikeal L. Roose

New potential citrus germplasm accessions may be received as seed rather than budwood, thereby reducing phytosanitary risks. However, trueness-to-type may be an issue with seed materials because many varieties produce both apomictic (nucellar) and sexual (zygotic) embryos and most citrus is fairly heterozygous. To identify nucellar seedlings of polyembryonic types and to retain these as representing the type, we screened 1340 seedlings from 88 seed sources for markers amplified with two inter-simple sequence repeat (ISSR) primers. Sixteen seed sources produced no seedlings classified as being of nucellar origin. Among the remaining seed sources, seedlings classed as nucellar were identified for potential addition to the collection. In 37 accessions, both nucellar and zygotic seedlings were detected, and in some cases both types were retained. Inclusion of established accessions of the same cultivar group in the analysis allowed an initial assessment of similarity to existing accessions. This technique improved the efficiency of acquiring new germplasm of polyembryonic types by seed. The method identifies those seed sources that produce few or no nucellar seedlings, but it is not useful for determining which seedlings of monoembryonic types should be retained in collections.

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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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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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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.

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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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M. Dolores Loureiro, M. Carmen Martínez, Jean-Michel Boursiquot, and Patrice This

`Albariño' (Vitis vinifera L.) is an important grape cultivar in Spain, morphologically diverse but subject to much misnaming. The objectives of the present work were to correct some of the more common misnamings concerning `Albariño' and to evaluate the genetic variability within this cultivar by analyzing DNA polymorphisms using randomly amplified polymorphic DNA (RAPD) markers and microsatellite techniques. Several accessions of `Albariño' (16 accessions from Misión Biológica de Galicia, one accession from El Encin, one accession from Rancho de la Merced), related cultivars (`Alvarinho', `Caíño blanco', `Cainho branco', `Loureiro'), and cultivars presumably identical to misnomers (`Savagnin blanc' and `Gewürztraminer') were analyzed using 20 RAPD markers and six microsatellite loci. Both techniques revealed polymorphism among `Albariño', `Caíño blanco', `Albariño' from Rancho de la Merced and `Loureiro'. No polymorphism was detected among the 16 `Albariño' accessions from Galicia, the `Albariño' accession from El Encin and `Alvarinho', nor among the `Albariño' accession from Rancho de la Merced, `Savagnin blanc' and `Gewürztraminer', nor between `Caíño blanco' and `Cainho branco'. These results enabled us to clarify the main misnomers concerning these cultivars. The absence of polymorphism among the true `Albariño' accessions did not allow the detection of any clonal variation. The suitability of both techniques for defining the cultivar level for grapevine is discussed.

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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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Martha Dávila, Dermot Coyne, Shree Singh, and Guenhwa Jung

The genes involved in F1 seedling abnormal development and lethality in inter-gene pool crosses have been designated as Dl1 (MesoAmerican=MA) and Dl2 (Andean=A) (Shii et al., 1980, J. Hered. 71:218–222). The different degrees of leaf crippling (C) in segregating populations of crosses was due to the interaction between the Dl1 or Dl2 loci, growing environment, and the lcr allele (Singh and Molina, 1996, J. Hered., In press). The objective was to identify RAPD markers linked to the genes for crippling (lcr) and seedling lethality (Dl) using the bulked segregation analysis procedure for F2 of MA × A crosses. Crosses were made between C lines, FB 10413-24-2, WA 7807-305, and TY 5578-220 and normal (N) parents and tester stocks for Dl1 and Dl2 genes. The F2 FB 10413-24-2 × Carioca segregated 13 N:3C. F3 families segregated 3N:1C. RAPD marker OPB-10 was linked to Lcr at 31.2 cM. F3 families segregated 1N:3C. RAPD marker OPO16 was linked to Dl1 at 27 cM. The F2 WA-7807-305 × Rio Tibagi segregated 3N:1C. RAPD marker OPS-03 was linked to Lcr at 32.6 cM.

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Hong Y. Yang, Schuyler S. Korban, Jutta Kruger, and Hanna Schmidt

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene Vf. Our major objective is to identify RAPD markers linked to the Vf gene. The approach in this paper is based on the introgression of the Vf gene from M. floribunda into commercial cultivars. Almost 200 random sequence decamer-primers have been used to screen a pair of bulked samples and the donor parent M. floribunda clone 821 for markers linked to the Vf gene conferring resistance to apple scab. A single primer has been identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars have confirmed that this marker is linked to the scab-resistance gene Vf. OPK16/1300 has since been cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker have been synthesized and used to screen further M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. The sequence-specific primers have identified polymorphisms of OPK16/1300 based on the presence or absence of a single band.

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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.