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Eric Stafne, John Clark, and Kim Lewers

Molecular markers have been used previously to identify linkages to important traits of interest. In this study two marker types, randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR), were used to find molecular markers linked to two morphological traits in blackberry (Rubus L. subgenus Rubus). Thorniness and floricane fruiting are both qualitative, recessive traits that are inherited tetrasomically. A cross of `Prime-Jim'® × `Arapaho' was made to create a population that segregated for the two traits. A random sample of 98 plants from a population of 200 were assayed to find molecular markers that co-segregate with the two traits. Three putative markers were identified for the floricane fruiting trait (two SSRs and one RAPD; χ2 = 4.09 to 9.99, P < 0.001 to 0.043). Five potential RAPD markers were found for the thorny trait (χ2 = 3.88 to 10.23, P < 0.001 to 0.048). Identification of markers linked to these traits could potentially be useful in marker-assisted selection.

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Ryan Walker, Leonard Pike, Monica Menz, Kil Sun Yoo, and Sung Gil Kim

Onions suffer from severe inbreeding depression, which has prevented the creation of homozygous inbreds in breeding programs. The creation of doubled haploid (DH) lines in onion is an anomaly, and provides a unique opportunity to study both how these lines would perform in a breeding program and why they do not exhibit more inbreeding depression. Seven DH lines were developed at Texas A&M University from five different short-day F1 and F2 populations. Five equivalent conventional inbred lines, breeding lines developed from the same parents as the DH lines, have been identified from the onion-breeding program. The DH lines, the conventional inbred lines, and their parents have been analyzed using SSR and AFLP markers. SSR markers yielded a polymorphism rate of 55% vs. 3% with the AFLPs. Analysis has shown remnant heterozygosity both in the parental lines and in the conventional inbreds and has confirmed the uniformity of the DH lines. Genetic similarity estimates have been calculated using the molecular data to determine the suitability of comparing the yield heterosis of crosses between the conventional lines and the DH lines in a diallele analysis.

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Warren Lamboy, Christopher Alpha, Amy Szewc-McFadden, and Sharon Bleik

The cold-hardy Vitis (grape) collection at the USDA/ARS Plant Genetic Resources Unit in Geneva, N.Y., comprises ≈1300 accessions. While much of the collection has been evaluated for morphological and viticultural traits, little of it has been well-characterized genetically. Lack of genetic information hampers the identification of accessions, the determination of genetic relationships among them, the evaluation of potential new accessions, and the construction of a core subset of the collection. Because simple sequence repeat DNA polymorphisms (SSRs or microsatellites) have already been proven to be useful genetic markers in Vitis vinifera (non-cold-hardy wine, raisin, and table grapes), our research focuses on the use of the markers both for the identification (“fingerprinting”) of species, hybrids, subspecies, cultivars (varieties), and accessions of cold-hardy Vitis, and for the determination of genetic relationships between these taxa. Our latest research results in this area will be presented.

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Kentaro Kitahara, Shogo Matsumoto, Toshiya Yamamoto, Junichi Soejima, Tetsuya Kimura, Hiromitsu Komatsu, and Kazuyuki Abe

We examined the genetic diversity and relatedness among apple (Malus ×domestica Borkh.) cultivars in Japan. The 42 apple cultivars, including major cultivars in Japan, were divided into five groups based on SSR genotypes. Most economically important cultivars belong in three groups: Fuji-Delicious, Golden Delicious, and Jonathan groups, and their genetic backgrounds seemed to be narrow. We also investigated the parent-offspring relationships of nine apple cultivars. `Jonathan', `Fuji', and `Rero 11' were identified as the respective paternal parents of three cultivars described as having unknown paternal parents (i.e., `Akagi', `Ambitious', and `Hokuto'). `Starking Delicious', `Senshu', and `Golden Delicious', rather than `Ralls Janet', `Hatsuaki', and `Indo', seemed to be the paternal parents of `Kinsei', `Kiou', and `Mellow', respectively. `Carolina Red June' was excluded as a paternal parent of `Ranzan'. Both attributed parents of `Scarlet' (`Akane' and `Starking Delicious') were excluded, and it was suggested that `Fuji' was used as either a maternal or a paternal parent of `Scarlet'. `Jonathan' rather than `McIntosh' seems to be a maternal parent of `Yukari'.

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A. Levi, C.E. Thomas, T. Trebitsh, A. Salman, J. King, J. Karalius, M. Newman, O.U.K. Reddy, Y. Xu, and X. Zhang

Seventy-one amplified fragment length polymorphism (AFLP), 93 sequence related amplified polymorphism (SRAP), and 14 simple sequence repeat (SSR) markers were used to extend an initial genetic linkage map for watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai]. The initial map was based on 151 randomly amplified polymorphic DNA (RAPD) and 30 and inter-simple sequence repeat (ISSR) markers. A testcross population previously used for mapping of RAPD and ISSR markers was used in this study: {plant accession Griffin 14113 [C. lanatus var. citroide (L.H. Bailey) Mansf.] × the watermelon cultivar New Hampshire Midget (C. lanatus var. lanatus)} × PI 386015 [C. colocynthis (L.) Schrad.]. The linkage map contains 360 DNA markers distributed on 19 linkage groups, and covers a genetic distance of 1976 cM with an average distance of 5.8 cM between two markers. A genomic DNA clone representing 1-amino-cyclopropane-1-carboxylic acid (ACC-) synthase gene, involved in ethylene biosynthesis, was also mapped. As in previous mapping studies for watermelon, a large number of AFLP and SRAP markers were skewed away from the 1:1 segregation ratio, and had to be excluded from the final mapping analysis. The stringent mapping criteria (JoinMap 3.0 mapping program) produced linkage groups with marker order consistent with those reported in previous mapping study for watermelon.

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Hongmei Ma and Margaret Pooler

Ornamental flowering cherry trees (Prunus species) are popular landscape plants that are used in residential and commercial landscapes throughout most temperate regions of the world. Most of the flowering cherry trees planted in the United States represent relatively few species. The U.S. National Arboretum has an ongoing breeding program aimed at broadening this base by developing new cultivars of ornamental cherry with disease and pest resistance, tolerance to environmental stresses, and superior ornamental characteristics. Knowledge of the genetic relationships among species would be useful in breeding and germplasm conservation efforts. However, the taxonomy of flowering cherry species and cultivars is complicated by differences in ploidy levels and intercrossing among species. We have used simple sequence repeat (SSR) markers developed for other Prunus species to screen a diverse collection of over 200 ornamental cherry genotypes representing 70 taxa in order to determine the genetic relationships among species, cultivars, and accessions. Data were generated from 9–12 primer pairs using an automated DNA genetic analyzer (ABI3770), and subjected to UPGMA cluster analysis. Extremely high levels of polymorphism were exhibited among the materials studied, thus indicating that ornamental flowering cherry germplasm has substantial inherent genetic diversity. This information, combined with traditional morphological characteristics, will be useful in determining genetic relationships among accessions in our collection and for predicting crossability of taxa.

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Anna Hale and Mark W. Farnham

Private and public vegetable breeders are interested in using current and emerging PCR-based marker systems in their respective improvement programs. However, before new systems are employed to replace existing ones, the new systems must prove to be efficient and cost-effective alternatives. Sequence related amplified polymorphisms (SRAPs), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) were compared for their ability to differentiate individuals of a diverse group of 24 elite broccoli (Brassica oleracea L. italica) inbreds. Genomic DNA was assayed using 24 AFLP, 24 SRAP, and 44 SSR primer pairs. In this assessment, SSRs produced an average of only two bands per primer, with 25% of these bands being monomorphic, and the remaining bands detecting very few differences among the inbreds. Although the AFLP method resulted in a lower rate (63%) of polymorphism than the SSRs, it produced about 20 bands per primer. SRAPs produced an average of 14 bands per primer, with 82% of these bands being polymorphic. Since AFLP and SRAP markers had a higher multiplex ratio and SSRs were frequently monomorphic, AFLP and SRAPs were more effective in differentiating the elite broccoli inbreds examined in this study. Similarity matrices were generated from the AFLP and SRAP data, and resulting dendographs were compared.

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Warren F. Lamboy and Christopher G. Alpha

Curators of plant genetic resources collections must preserve germplasm possessing known useful characteristics as well as material displaying general genetic diversity. In order to ensure that both types of germplasm are included in a collection, germplasm curators require three fundamental types of information about each accession: taxonomic identity, genetic identity, and genetic relationship. Because simple sequence repeat DNA fragments (SSRs) have been successfully used to determine the genetic identity of grape clones, we conducted a study to determine if SSRs would supply all three types of information for the accessions in the cold-hardy Vitis (grape) germplasm collection. SSR fragments were amplified at six different loci for 23 accessions of cold-hardy grape spanning the range of species diversity in the collection. The minimum number of different alleles found at a locus was 9; the maximum was 26. Heterozygosity values ranged between 0.565 and 0.783, while gene diversity values were in the range 0.785 to 0.944. Two hundred fifty-two pairs of plants out of a possible 253 could be distinguished by their SSR profiles. Nei's genetic identities were computed between all pairs of plants and used in a UPGMA cluster analysis. The relationships obtained did not correspond well to expected relationships based on geography and taxonomy. Four species of grapes were represented by two or more accessions in this study. No DNA fragments found at these six loci served to unambiguously distinguish one species from another. Thus, SSR fragments from the six loci studied were useful in determining genetic identity of accessions, but were not helpful in determining genetic relationships or taxonomic identities. We are searching for additional loci that are informative for these types of information. Meanwhile we highly recommend SSRs for determining genetic identity in germplasm resources collections.

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Minou Hemmat, Norman F. Weeden, Patrick J. Conner, and Susan K. Brown

The columnar mutation `Wijcik McIntosh' has attracted much attention because of its compact growth habit, which is compatible with high-density plantings. Using bulked segregant analysis, we identified several randomly amplified polymorphic DNA (RAPD) markers that displayed a close linkage with the columnar locus (Co). The RAPD marker that displayed the closest linkage was end sequenced to develop a sequence tagged site for rapidly screening segregating populations. A simple sequence repeat (SSR) of (GA)17 was identified within the DNA fragment. Four allelic forms, including an apparent null allele, could be distinguished among the cultivars tested. The null allele displayed close linkage with Co in two progenies, and we used this marker to identify the location of the gene on the apple linkage map.

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Riaz Ahmad, Louise Ferguson, and Stephen M. Southwick

A genomic DNA library enriched for dinucleotide (CT)n and (CA)n and trinucleotide (CTT)n microsatellite motifs has been developed from `Kerman' pistachio (Pistacia vera L.). The enrichment method based on magnetic or biotin capture of repetitive sequences from restricted genomic DNA revealed an abundance of simple sequence repeats (SSRs) in the pistachio genome which were used for marker development. After an enrichment protocol, about 64% of the clones contained (CT)n repeats while 59% contained (CA)n for CT and CA enriched libraries, respectively. In the (CT)n enriched library, compound sequences were 45% while for (CA)n it was 13.5%. In both dinucleotide enriched libraries, about 80% of the clones having microsatellites have a repeat length in the range of 10 to 30 units. A library enriched for trinucleotide (CTT)n contained <19% of the clones with (CTT)n repeats. Of the clones that contained microsatellites, 62% had sufficient flanking sequence for primer design. An initial set of 25 pairs of primers was designed, out of which 14 pairs amplified cleanly and produced an easily interpretable PCR product in the commercially important American, Iranian, Turkish, and Syrian pistachio cultivars. The efficient DNA extraction method developed for pistachio kernels and shells (roasted and nonroasted) yielded DNA of sufficient quality to use PCR to create DNA fingerprints. In total, 46 alleles were identified by 14 primer pairs and a dendrogram was constructed on the basis of that information. The SSR markers distinguished most of the tested cultivars from their unique DNA fingerprint. An UPGMA cluster analysis placed most of the Iranian samples in one group while the Syrian samples were the most diverse and did not constitute a single distinct group. The maximum number of cultivar specific markers were found in `Kerman'(4), the current industry standard in the United States, and the Syrian cultivar Jalab (5). The technique of using extracted DNA from pistachio kernal or shell coupled with the appropriate marker system developed here, can be used for analyses and measurement of trueness to type.