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Darush Struss, Riaz Ahmad, Stephen M. Southwick, and Manuela Boritzki

Simple sequence repeats (SSRs) and amplified fragment-length polymorphisms (AFLPs) were used to evaluate sweet cherry (Prunus avium L.) cultivars using quality DNA extracted from fruit flesh and leaves. SSR markers were developed from a phage library using genomic DNA of the sweet cherry cultivar Valerij Tschkalov. Microsatellite containing clones were sequenced and 15 specific PCR primers were selected for identification of cultivars in sweet cherry and for cross-species amplification in Prunus. In total, 48 alleles were detected by 15 SSR primer pairs, with an average of 3.2 putative alleles per primer combination. The number of putative alleles ranged from one to five in the tested cherry cultivars. Forty polymorphic fragments were scored in the tested cherry cultivars by 15 SSRs. All sweet cherry cultivars were identified by SSRs from their unique fingerprints. We also demonstrated that the technique of using DNA from fruit flesh for analysis can be used to maintain product purity in the market place by comparing DNA fingerprints from 12 samples of `Bing' fruit collected from different grocery stores in the United States to that of a standard `Bing' cultivar. Results indicated that, with one exception, all `Bing'samples were similar to the standard. Amplification of more than 80% of the sweet cherry primer pairs in plum (P. salicina), apricot (P. armeniaca) and peach (P. persica L.) showed a congeneric relationship within Prunus species. A total of 63 (21%) polymorphic fragments were recorded in 15 sweet cherry cultivars using four EcoRI-MseI AFLP primer combinations. AFLP markers generated unique fingerprints for all sweet cherry cultivars. SSRs and AFLP polymorphic fragments were used to calculate a similarity matrix and to perform UPGMA cluster analysis. Most of the cultivars were grouped according to their pedigree. The SSR and AFLP molecular markers can be used for the grouping and identification of sweet cherry cultivars as a complement to pomological studies. The new SSRs developed here could be used in cherry as well as in other Prunus species for linkage mapping, evolutionary and taxonomic study.

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Amy K. Szewc-McFadden, Warren F. Lamboy, and James R. McFerson

To comprehend genetic identity and relatedness in Malus germplasm held in situ and ex situ, we are employing simple sequence repeat (SSR) DNA fragment information in combination with passport and horticultural data. SSRs offer certain advantages for characterizing large arrays of germplasm efficiently. They are abundantly dispersed throughout plant genomes and are exceedingly polymorphic. In addition, they can be PCR-amplified and detected by automated fluorescence-based technology. A size-fractionated DNA library of M. ×domestica cv Golden Delicious was screened to identify SSR loci. Eight loci were found to be reliably informative and were used to prepare locus-specific primer pairs. Characterization of the 75 M. ×domestica accessions included in the core subset of the USDA-ARS Malus germplasm collection revealed six of the eight loci were polymorphic within the array. The number of alleles per locus ranged from two to 21. Throughput was enhanced by multiplexing, allowing simultaneous use of two or three primer pairs. With improved genetic characterization of Malus germplasm, we intend to better develop and relate the core subset to the rest of the collection and to in situ Malus genetic resources. SSR markers appear to be an efficient and reliable tool to expedite this process.

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Yiqi Zhen, Zuozhou Li, Hongwen Huang, and Ying Wang

Forty-eight kiwifruit cultivars and selections, representing more than 90% of total world kiwifruit production, were investigated using nine SSR markers to establish genetic identities, and evaluate genetic diversity and relatedness. These nine SSRs were polymorphic and a total of 213 alleles were detected, resulting in a mean number of 23.7 alleles per locus, ranging from nine to 38 alleles. One hundred and thirty-three alleles were found to be common to both A. chinensis and A. deliciosa, while 33 and 36 were specific to A. chinensis and A. deliciosa, respectively. In addition, 34 alleles were specific to one single genotype and provided a set of valuable alleles for cultivar identification. A single SSR locus UDK 96-414 could differentiate all 48 genotypes except two presumable clones. Mean number of alleles per locus (A), percentage of polymorphic loci (P), and direct count heterozygosity (Ho) assessed for each genotype over all loci revealed considerable differences among these 48 genotypes. On average, A = 2.6, P = 89.4% and Ho = 0.546 were found in A. chinensis cultivars, while A = 3.5, P = 97.0% and Ho = 0.671 in A. deliciosa cultivars. Consensus fingerprint profiling using SSR markers is a useful and reliable method for establishing genetic identities of kiwifruit cultivars and selections. It also improves evaluation effectiveness of genetic diversity and relatedness compared to RAPD markers.

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Xinwang Wang, Robert N. Trigiano, Mark T. Windham, Renae DeVries, Timothy A. Rinehart, James M. Spiers, and Brain Scheffler

The genus Cornus consists of many species, of which C. florida, C. kousa, C. mas, and C. stolonifera are four main ornamental species in North America, Asia, and Europe. For example, over 200 cultivars of C. florida alone have been developed for the nursery industry. Microsatellite loci, or SSR, are useful markers for studying genetic diversity and for creating linkage maps of the various species. The objective of this study was to investigate the genetic diversity between these four Cornus species and eight hybrids. Evaulation of the diversity will be useful in assessing the selection pressure of breeders and/or genetic drift of these dogwood cultivars/lines. Fifteen SSR primer pairs were selected to examine 56 Cornus cultivars and/or lines of the four species and hybrids. The study included 28 C. florida cultivars and lines, 15 C. kousa cultivars and lines, four C. stolonifera cultivars, one cultivar of C. mass and eight hybrids between various Cornus species. An exceptionally high level of diversity was detected among the 56 entries in both the number and size range of SSR alleles. A total of 95 alleles with an average of 7.8 alleles per loci were detected among these 56 genotypes. These selected Cornus cultivars and/or lines could be clustered into four to six subgroups. Some Cornus species were integrated into other species groups, suggesting gene flow between species via the breeding or evolution. SSR markers can contribute to the exploitation of genetic diversity for existing Cornus germplasm. For further study, examination of more SSR loci could explain more completely the diversity among these Cornus cultivars and lines.

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E.J. Parks, J.W. Moyer, and J.H. Lyerly

Fluorescent amplified fragment length polymorphism (F-AFLP) and microsatellites (SSRs) were used to evaluate new guinea impatiens (Impatiens hawkeri W. Bull) cultivars. Ninety-five quality-selected polymorphic fragments from 10 F-AFLP+3 primer combinations were used to evaluate 100 cultivars representing a variety of colors, forms, and breeding programs. Jaccard similarities and unweighted pair-group method of the arithmetic average (UPGMA) clustering formed a dendrogram with three cultivar groups, to a large extent clustering the cultivars by breeder with a high cophenetic correlation coefficient. A small insert genomic library was created and 442 kb of new guinea impatiens sequence was screened for repetitive motifs, resulting in 14 microsatellite markers. A subset of 46 cultivars representing five commercial breeding companies and 11 cultivar series was selected for microsatellite analysis. Seven loci were polymorphic, with two to six alleles per locus. Although both methods were equally effective in distinguishing the cultivars from one another, the topologies of the dendrograms for the two methods were different. The topology of the AFLP dendrogram reflected possible relationships based on cultivar series and breeding company, while the SSR dendrogram did not. The objectives of this research were to develop and validate both F-AFLP and SSR methodologies for new guinea impatiens, identify markers that can be reliably used for fingerprinting, and create a database for future cultivar comparisons.

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Minou Hemmat, Norman F. Weeden, Frank S. Cheng, and S.K. Brown

The positions of over 50 SSR loci and other sequence tagged sites (STSs) have been located on the linkage maps of five apple cultivars (Rome Beauty, White Angel, Golden Delicious, Liberty, McIntosh) and two New York accessions. In most cases, the primers used produced single amplification products, permitting identification of homologous loci in the different cultivars and the precise alignment of the linkage maps generated for each. Based on this information, we present a general linkage map for apple with STS markers on each linkage group. The map consists of 17 linkage groups (equal to the haploid chromosome number for the species) with over 500 markers. The positions of several resistant gene analogues have been located on this linkage map. None of these sequences map near genes conferring resistance to scab or powdery mildew. SSR loci exhibited a tendency to cluster in certain regions of the linkage map. This clustering slightly reduces their effectiveness as genome markers for comparative mapping or germplasm diversity. However, the SSR markers definitely displayed a high level of polymorphism, making them particularly useful for genetic studies.

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Anna L. Hale, Mark W. Farnham, and Monica A. Menz

Breeders of cole crops (Brassica oleracea L.) have an interest in utilizing current and emerging PCR-based marker systems to differentiate elite germplasm. However, until efficiency and cost-effectiveness are determined, most breeders are hesitant to change methods. In this study, our goal was to compare simple sequence repeat (SSR), amplified fragment-length polymorphism (AFLP), and sequence-related amplified polymorphism (SRAP) marker systems for their effectiveness in differentiating a diverse population of 24 elite broccoli (B. oleracea Italica Group) inbreds. Published SSR primer sequences for Brassica L. species were used along with AFLP and SRAP primer combinations. Several SSR primers failed to amplify DNA in the broccoli population, but all AFLP and SRAP primer combinations produced multiple bands. Twenty-nine percent of the SSR primers were monomorphic, while most of the remaining primers detected only one or two differences among inbreds. AFLP and SRAP methods produced multiple differences per primer in almost every case. Phenetic analysis revealed that the type of marker affected the classification of the genotypes. All three marker systems were able to successfully differentiate between the 24 elite inbreds, however, AFLPs and SRAPs were more efficient, making them better alternatives than SSRs over other established methods for fingerprinting B. oleracea inbreds.

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Minou Hemmat, Norman F. Weeden, and Susan K. Brown

We mapped DNA polymorphisms generated by 41 sets of Simple Sequence Repeat (SSR) primers, developed independently in four laboratories. All primer sets gave polymorphisms that could be located on our `White Angel' x `Rome Beauty' map for apple [Malus sylvestris (L.) Mill. Var. domestica (Borkh.) Mansf.]. The SSR primers were used to identify homologous linkage groups in `Wijcik McIntosh', NY 75441-58, `Golden Delicious', and `Liberty' cultivars for which relatively complete linkage maps have been constructed from isozyme and Random Amplified Polymorphic DNA (RAPD) markers. In several instances, two or more SSRs were syntenic, and except for an apparent translocation involving linkage group (LG) 6, these linkages were conserved throughout the six maps. Twenty-four SSR primers were consistently polymorphic, and these are recommended as standard anchor markers for apple maps. Experiments on a pear (Pyrus communis L.) population indicated that many of the apple SSRs would be useful for mapping in pear. However some of the primers produced fragments in pear significantly different in size than those in apple.

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Stan C. Hokanson, Amy K. Szewc-McFadden, Warren F. Lamboy, and James R. McFerson

A diverse collection of 133 Malus species and hybrids from the USDA Plant Genetic Resources Unit's core subset collection was screened with five simple sequence repeat (SSR) primer pairs in order to determine genetic identities and overall levels of genetic variation. The number of amplification products (alleles) per locus (primer pair) in this collection ranged from 6 to 39, with some genotypes showing complex banding patterns of up to four products per locus, suggesting that duplication events may have occurred within the genome. Five primer sets unequivocally differentiated all but 10 pairs of genotypes in the collection, with seven of these 10 being pairs of the same species. Within three of the species holdings surveyed, M. honanensis, M. sargentii, and M. sikkimensis, no genetic variation was revealed with the SSR markers. The discrimination power for the combined loci in this collection was nearly one, which indicates that the likelihood of two genetically different accessions sharing the same alleles at all the loci included in this study would be nearly impossible. Coupled with results from a previous survey of M. × domestica accessions, this finding suggests that with five SSR primer pairs, the majority of the Malus holdings could be assigned a unique fingerprint identity. The average direct count heterozygosity over all loci was 0.620, ranging in value from 0.293 to 0.871 over individual loci. These heterozygosity counts will be compared with a survey of naturally occurring M. sieversii to determine whether current repository holdings are representative of the overall levels of diversity occurring in Malus. Information generated with this study, coupled with passport and horticultural data will inform curatorial decisions regarding deaccessioning of duplicate holdings and plans for future germplasm collections.

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Zhi-Hong Gao, Zhi-Jun Shen, Zhen-Hai Han, Jing-Gui Fang, Yu-Ming Zhang, and Zhen Zhang

Sequencing amplification fragments produced using simple-sequence repeat (SSR) primer pairs pchgms2 and UDP96008 in `Dayezhugan' japanese apricot showed that SSRs obtained included a microsatellite locus originally identified in peach. The microsatellite sequence homogeneity between UDP96008 in japanese apricot in this study and UDP96008 in the peach in GenBank was 98%. Twenty-four japanese apricot genotypes originating in diverse geographic areas had been identified with 14 SSR primer pairs developed in different species of Prunus. In total, 129 alleles were obtained and per primer pairs detected 2.5 alleles on the average. The results from cluster analysis showed that the genetic distance between `Nanhong' and `Zhonghong' was the closest, and cultivars from China and from Japan could not be separated completely.