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Hongwen Huang, Desmond R. Layne and R. Neal Peterson

The utility of isozyme phenotypes for identifying and determining genetic variation in pawpaw cultivars was studied using isoelectric focusing in thin-layer polyacrylamide gels. Based on a sample of 32 clones (cultivars and advanced selections) and 23 enzyme systems, 7 enzymes were found to be polymorphic, involving 9 polymorphic loci [acid phosphatase (ACP), dihydrolipoamide dehydrogenase (DDH), malic enzyme (ME), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), peroxidase (PRX), and shikimate dehydrogenase (SKD)]. Altogether these 9 loci and 32 clones yielded 28 multi-locus isozymic phenotypes useful for cultivar identification; 24 of the 32 clones were uniquely identified. The allozyme variation in these clones has the average of other long-lived woody perennials of widespread geographic range in temperate regions with insect-pollinated outcrossing breeding systems, secondary asexual reproduction, and animal-dispersed seed. Genetic differentiation among these pawpaw clones, measured by Nei's distance, D, was substantial: 496 pairwise comparisons of genetic distance among the 32 clones indicated that they differed on average of D = 0.068 ± 0.04 and ranged from 0 to 0.188. Cluster analysis (UPGMA) produced a most likely division of the 32 clones into 7 groups; however, these groups did not conform to known pedigree relations. Additional polymorphic enzymes are needed for accurate allozyme-based genetic discrimination.

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Zhanao Deng and Brent K. Harbaugh

The ornamental value of caladium (Caladium ×hortulanum Birdsey) depends primarily on leaf characteristics, including leaf shape and main vein color. Caladium leaf shapes are closely associated with plant growth habit, stress tolerance, and tuber yield; leaf main vein colors are often used for cultivar identification. Thirty-eight crosses were made among 10 cultivars and two breeding lines; their progeny were analyzed to understand the inheritance of leaf shape and main vein color and to determine if there is a genetic linkage between these two traits. Results showed that a single locus with three alleles determined the main vein color in caladium. The locus was designated as V, with alleles V r, V w, and V g for red, white, and green main veins, respectively. The white vein allele was dominant over the green vein allele, but it was recessive to the red vein allele, which was dominant over both white and green vein alleles; thus the dominance order of the alleles is V r > V w > V g. Segregation data indicated that four major red-veined cultivars were heterozygous with the genotype Vr V g, and that one white-veined cultivar was homozygous and one other white-veined cultivar and one breeding line were heterozygous. The observed segregation data confirmed that the three leaf shapes in caladium were controlled by two co-dominant alleles at one locus, designated as F and f, for fancy and strap leaves, respectively. The skewedness of leaf shape segregation in some of the crosses implied the existence of other factors that might contribute to the formation of leaf shape. Contingency chi-square tests for independence revealed that caladium leaf shape and main vein color were inherited independently. The chi-square tests for goodness-of-fit indicated that the five observed segregation patterns for leaf shape and main vein color fit well to the expected ratio assuming that two co-dominant and three dominant/recessive alleles control leaf shape and main vein color and they are inherited independently.

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L.H. Zhang, D.H. Byrne, R.E. Ballard and S. Rajapakse

Microsatellite or simple sequence repeat (SSR) markers were developed from Rosa wichurana Crépin to combine two previously constructed tetraploid rose (Rosa hybrida L.) genetic maps. To isolate SSR-containing sequences from rose a small-insert genomic library was constructed from diploid Rosa wichurana and screened with several SSR probes. Specific primers were designed for 43 unique SSR regions, of which 30 primer pairs gave rise to clear PCR products. Seventeen SSR primer pairs (57%) produced polymorphism in the tetraploid rose 90-69 mapping family. These markers were incorporated into existing maps of the parents 86-7 and 82-1134, which were constructed primarily with AFLP markers. The current map of the male parent, amphidiploid 86-7, consists of 286 markers assigned to 14 linkage groups and covering 770 cm. The map of the female tetraploid parent, 82-1134, consists of 256 markers assigned to 20 linkage groups and covering 920 cm. Nineteen rose SSR loci were mapped on the 86-7 map and 11 on the 82-1134 map. Several homeologous linkage groups within maps were identified based on SSR markers. In addition, some of the SSR markers provided anchoring points between the two parental maps. SSR markers were also useful for joining small linkage groups. Based on shared SSR markers, consensus orders for four rose linkage groups between parental maps were generated. Microsatellite markers developed in this study will provide valuable tools for many aspects of rose research including future consolidation of diploid and tetraploid rose genetic linkage maps, genetic, phylogenetic and population analyses, cultivar identification, and marker-assisted selection.

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Barbara S. Gilmore, Nahla V. Bassil, Danny L. Barney, Brian J. Knaus and Kim E. Hummer

Identifying and evaluating genetic diversity of culinary rhubarb (Rheum ×rhababarum) cultivars using morphological characteristics is challenging given the existence of synonyms and nomenclatural inconsistencies. Some cultivars with similar names are morphologically different, and seedlings may grow and become associated with the parental name. Morphological traits of one cultivar may vary when measured under different environmental conditions. Molecular markers are consistent for unique genotypes across environments and provide genetic fingerprints to assist in resolving identity issues. Microsatellite repeats, also called simple sequence repeats (SSRs), are commonly used for fingerprinting fruit and nut crops, but only 10 SSRs have previously been reported in rhubarb. The objectives of this study were to use short-read DNA sequences to develop new di-nucleotide-containing SSR markers for rhubarb and to determine if the markers were useful for cultivar identification. A total of 97 new SSR primer pairs were designed from the short-read DNA sequences. The amplification success rate of these SSRs was 77%, whereas polymorphism of those reached 76% in a test panel of four or eight rhubarb individuals. From the 57 potentially polymorphic primer pairs obtained, 25 SSRs were evaluated in 58 Rheum accessions preserved in the U.S. Department of Agriculture, National Plant Germplasm System. The primer pairs generated 314 fragments with an average of 12.6 fragments per pair. The clustering of many accessions in well-supported groups supported previous findings based on amplified fragment length polymorphisms (AFLPs). Cluster analysis, using the proportion of shared allele distance among the 25 SSRs, distinguished each of the 58 accessions including individuals that had similar names or the same name. Accessions that grouped in well-supported clusters previously belonged to similar clusters with high bootstrap support based on AFLP. In summary, our technique of mining short-read sequencing data was successful in identifying 97 di-nucleotide-containing SSR sequences. Of those tested, the 25 most polymorphic and easy-to-score primer pairs proved useful in fingerprinting rhubarb cultivars. We recommend the use of short-read sequencing for the development of SSR markers in the identification of horticultural crops.

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Marie-José Côté and Lisa Leduc

accurate identification becomes difficult. DNA fingerprinting techniques have been proven to be reliable for cultivar identification because the markers are not influenced by environmental factors, life stage, or type of plant tissue analyzed. The choice

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Kang Hee Cho, Jung Ho Noh, Seo Jun Park, Se Hee Kim, Dae-Hyun Kim and Jae An Chun

, 180 distinct markers were obtained from the RAPD analysis. The results obtained for each random primer are summarized in Table 2 . Unambiguous amplified DNA bands were carefully chosen and scored for cultivar identification to ensure the absence of

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Luis Rallo, Diego Barranco, Raúl de la Rosa and Lorenzo León

locations in Spain. Description Morphological description was carried out according to Barranco et al. (2000) by using a total of 25 characters of the tree, leaf, fruit, and endocarp. Cultivar identification by molecular markers was performed with

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Joseph C. Kuhl and Veronica L. DeBoer

information about cultivars is missing or never recorded, making cultivar identification difficult and nearly impossible to authenticate. Cultivar identification has primarily relied on morphology; however, such traits are influenced by environmental factors

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Aydin Uzun, Osman Gulsen, Gucer Kafa and Ubeyit Seday

number of seedless lemons varying in adaptive potential and fruit characteristics. In addition, new seedless cultivars should be tested under different environmental conditions for their performances. One major consideration from the view of cultivar

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Zahra Noormohammadi, Mehdi Hosseini-Mazinani, Isabel Trujillo, Luis Rallo, Angjelina Belaj and Majid Sadeghizadeh

understanding of the level and distribution of genetic diversity in olive cultivars, which may be of use in cultivar identification. In recent years, molecular markers such as isozymes ( Trujillo et al., 1995 ) randomly amplified polymorphic DNA (RAPD) ( Belaj