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Vance Baird

Prunus, which includes peach/nectarine, almond, apricot, cherry, and plum, is a large and economically important genus in the family Rosaceae. The size and long generation time of these tree crops have hampered improvement through classical breeding and long-term selection programs. With the advent of DNA-based molecular diagnostics, an exciting era in germplasm improvement has dawned. Efforts are underway, notably in the United States (e.g., California, Michigan, North Carolina, and South Carolina) and the European Community (e.g., England, France, Italy, and Spain), to apply the tools of molecular mapping and marker-assisted selection to this important genus. The objective of these projects is to develop molecular linkage maps of sufficient marker density to tag phenotypic trait loci of agronomic importance. These include traits controlled by single genes (e.g., flower color, compatibility, flesh color, pest resistance), as well as more-complex, quantitative traits (e.g., cold hardiness, tree architecture, sugar content). An immediate outcome of these mapping efforts has been the development of DNA “fingerprints,” allowing for the discrimination of cultivars—both scion and rootstock. The maps will be used by breeders and molecular biologists to monitor gene introgression from wild species into elite lines, for marker-assisted selection of desired trait combinations, and for map-based cloning of specific genes. The molecular markers used in these mapping projects include RFLPs, RAPDs, and microsatellites. Each has their appropriate applications and advantages depending upon the resources at hand and the project's specific goals.

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Riaz Ahmad, Dan Potter, and Stephen M. Southwick

Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) molecular markers were evaluated for detecting intraspecific variation in 38 commercially important peach and nectarine (Prunus persica) cultivars. Out of the 20 SSR primer pairs 17 were previously developed in sweet cherry and three in peach. The number of putative alleles revealed by SSR primer pairs ranged from one to five showing a low level of genetic variability among these cultivars. The average number of alleles per locus was 2.2. About 76% of cherry primers produced amplification products in peach and nectarine, showing a congeneric relationship within Prunus species. Only nine cultivars out of the 38 cultivars could be uniquely identified by the SSR markers. For SRAP, the number of fragments produced was highly variable, ranging from 10 to 33 with an average of 21.8 per primer combination. Ten primer combinations resulted in 49 polymorphic fragments in this closely related set of peaches and nectarines. Thirty out of the 38 peach and nectarine cultivars were identified by unique SRAP fingerprints. UPGMA Cluster analysis based on the SSR and SRAP polymorphic fragments was performed; the relationships inferred are discussed with reference to the pomological characteristics and pedigree of these cultivars. The results indicated that SSR and SRAP markers can be used to distinguish the genetically very close peach and nectarine cultivars as a complement to traditional pomological studies. However, for fingerprinting, SRAP markers appear to be much more effective, quicker and less expensive to develop than are SSR markers.

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Mikel R. Stevens, Shawn A. Chrisensen, Ammon B. Marshall, JoLynn J. Stevens, Peter Wenzl, Eric Hunter, Jason Carling, and Andrzej Killian

Recently, a technology known as DArT (diversity array technology) has been developed to increase throughput in marker assisted selection (MAS). DArT utilizes microarray technology as a method to potentially compare thousands of molecular markers in one test to a single DNA sample. We used DArT on two sets of interspecific tomato [Solanum lycopersicum (Fla 7613) × S. pennellii (LA 716 or LA 2963)] segregating populations (BC, F2, and F1). We compared over 300 segregating plants to 3840 random tomato genomic fragments. After the 3840 markers were prepared, it took about 2 weeks of laboratory time to perform the experiments. With experience, this time can be reduced. We identified a total of 654 polymorphic markers usable for developing a DArT tomato genetic map. Depending on the particular cross, 13 to 17 linkage groups were identified (LOD 3) per population. Most recently, the amplified polymorphic DNA (AFLP) technique has been used for rapid genetic mapping of large numbers of anonymous genomic fragments. Besides the additional effort and reagents using AFLPs compared to DArT, a desired AFLP polymorphic band is often difficult to clone and process into a PCR based marker, whereas in DArT all markers are already cloned and immediately available for such experiments. A drawback to DArT is that it requires specialized software and equipment and is technically demanding. However, once the equipment and software are secured, techniques are optimized, and segregating populations developed, marker throughput is increased by orders of magnitude. Although challenging, the application of DArT can dramatically increase MAS throughput, thus facilitating quantitative trait and saturated mapping research.

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Jericó J. Bello-Bello, Lourdes G. Iglesias-Andreu, Susana A. Avilés-Viñas, Eunice Gómez-Uc, Adriana Canto-Flick, and Nancy Santana-Buzzy

repeat (SSR), restriction fragment length polymorphism, and amplified fragment length polymorphism. The use of these molecular markers to detect variations at genomic DNA level in plant has been clearly documented ( Andreev et al., 2005 ; Gernand et al

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

. Genetic diversity (H) of Nei (1973) and Shannon index ( S ) ( Lewontin, 1972 ) were used to summarize the data for molecular markers, and their standard deviations (SD) were indicated. The percentage of polymorphisms (Pp) was given as number of

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

below 0.73. Similar work has been reported in other ornamental plants and confirmed the value of molecular marker analysis in assessing genetic diversity or understanding genetic relationships among cultivars and species ( Ahmad et al., 2006 ; Carr et

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Patrick Conner, Joann Conner, Paige Catotti, Jennifer Lewter, John R. Clark, and Luiz A. Biasi

University of Georgia (U.S.A) breeding program Acta Hort. 1046 303 307 Dalbó, M.A. Ye, G.N. Weeden, N.F. Steinkellner, H. Sefc, K.M. Reisch, B.I. 2000 A gene controlling sex in grapevines placed on a molecular marker-based genetic map Genome 43 333 340

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K. Ikeda, A. Watari, K. Ushijima, H. Yamane, N.R. Hauck, A.F. Iezzoni, and R. Tao

S4′ is a pollen-part mutant in sweet cherry (Prunus avium L.) that is extensively used to develop self-compatible cultivars. The S4′-haplotype is known to have a functional stylar component and a nonfunctional pollen component. The pollen component in sweet cherry necessary for the specificity of the pollen reaction is believed to be an S-haplotype specific F-box protein gene, called SFB. This study describes two molecular markers that distinguish between SFB4 and SFB4′ by taking advantage of a four base pair deletion in the mutant allele. The resulting polymerase chain reaction (PCR) products can either be separated directly on a polyacrylamide gel or they can be subjected to restriction enzyme digestion and the different sized products can be visualized on an agarose gel. The latter technique utilizes restriction sites created in the PCR products from the SFB4′ allele, but not the SFB4 allele. Because the primer sets created differential restriction sites, these primer sets were termed dCAPS (derived cleaved amplified polymorphism sequence) markers. These molecular assays can be used to verify self-compatibility conferred by the S4′-haplotype.

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N. Mutlu, D.P. Coyne, S.O. Park, and J.R. Steadman

Common bacterial blight (CBB) in common bean (Phaseolus vulgaris L.), caused by Xanthomonas campestris pv. phaseoli (Xcp), reduces bean yields and quality throughout the world. Pinto `Chase' is a high-yielding variety with moderate resistance to Xcp derived from great northern Nebraska #1 selection 27, whose resistance is derived from an unknown tepary (P. acutifolius) bean source. XAN-159 is a black mottled small seeded breeding line with different genes for high resistance to Xcp derived from a different tepary source (PI 319443). Our objective was to pyramid different genes for Xcp resistance from the donor parent XAN-159 into the rust-resistant recurrent parent Pinto `Chase' using the classical back-cross breeding method with confirmation of resistance using RAPD molecular markers. Resistance was confirmed in some BC2F2 generation plants. Seven RAPD markers and the V locus (flower color) previously identified were confirmed in the BC1 and BC2 populations. Smaller seed size, purple flower color, and black mottled seed coat color were coinherited with resistance to Xcp. However, a recombinant plant with enhanced CBB resistance and moderate-sized pinto seed was identified. Backcross breeding is being continued.

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Tera M. Bonney, Shawn P. Brown, Snake C. Jones, Kirk W. Pomper, and Robert L. Geneve

The pawpaw [Asimina triloba (L.) Dunal] is a native plant found mainly in the southeastern and eastern United States, and its fruit has great potential as a new high-value crop in these regions. Although there are ≈45 named pawpaw cultivars, breeding for improvement of specific traits, such as fruit size and quality, is desirable. Our long-term goal is to utilize molecular marker systems to identify markers that can be used for germplasm diversity analyses and for the construction of a molecular genetic map, where markers are correlated with desirable pawpaw traits. The objective of this study was to identify random amplified polymorphic DNA (RAPD) markers that segregate in a simple Mendelian fashion in a controlled A. triloba cross. DNA was extracted from young leaves collected from field-planted parents and 20 progeny of the cross 1-7 × 2-54. The DNA extraction method used gave acceptable yields of ≈7 μg·g-1 of leaf tissue. Additionally, sample 260/280 ratios were ≈1.4, which indicated that the DNA was of high enough purity to be subjected to the RAPD methodology. Screening of 10-base oligonucleotide RAPD primers with template DNA from the parents and progeny of the cross has begun. We have identified two markers using Operon primer B-07 at 1.1 and 0.9 kb that segregate in a simple Mendelian fashion in progeny of the 1-7 × 2-54 cross. Other primers and controlled crosses will also be screened.