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Liwang Liu, Guang Liu, Yiqin Gong, Wenhao Dai, Yan Wang, Fanmin Yu, and Yunying Ren

from both parents, identification of male and female parent-specific markers will allow differentiation of true hybrids from selfed parental lines and outcrossed lines. Molecular markers, such as RAPD, ISSR, SSR, SRAP, AFLP (amplified fragment length

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Aoxue Wang, Fanjuan Meng, Xiangyang Xu, Yong Wang, and Jingfu Li

the demand for controlling leaf mold disease on tomatoes in these provinces. We developed molecular markers to the Cf-6 gene by the means of rapid amplification of polymorphic DNA (RAPD) and simple sequence repeat (SSR). These two methods, based on

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Maomao Ding, Ke Wang, Wenting Wang, Miaojin Chen, Dajun Wu, Changjie Xu, and Kunsong Chen

cultivars has instead been achieved by using various molecular makers. For example, using restriction fragment length polymorphism and random amplified polymorphic DNA ( Chaparro et al., 1994 ; Quarta et al., 1998 ; Rajapakse et al., 1995 ). SSR markers

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Tim Rinehart, Cecil Pounders, and Brian Scheffler

Crapemyrtles (Lagerstroemia) are deciduous shrubs or trees with prolific summer flowers. Their popularity is due in large part to low maintenance requirements in sunny climates, wide range of growth habits, disease resistance, and bark characteristics, as well as having a long flowering period (up to 120 days). Once well-established, they are extremely tolerant to heat and drought. Lagerstroemia was first introduced to the southern U.S. from southeast Asia more than 150 years ago, and is comprised of at least 80 known species. Most modern cultivars are L. indica and L. fauriei hybrids. L. speciosa is a tropical crapemyrtle with very large flowers, but lacks cold hardiness. It is a vigorous plant, but only when grown in Hardiness zones 9 or 10. We recently established microsatellite markers for Lagerstroemia and evaluated their utility for verifying interspecific hybrids. Here we verify F1 hybrids between L. indica `Tonto', `Red River', and L. speciosa. We also genotyped two commercially available L. speciosa hybrids. Currently, we are using crapemyrtle SSRs for cultivar identification and germplasm conservation. Future research includes marker-assisted breeding to produce powdery mildew and flea beetle resistant cultivars, as well as improved growth habit and fall foliage color.

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Kim S. Lewers*, Eric T. Stafne, John R. Clark, Courtney A. Weber, and Julie Graham

Some raspberry and blackberry breeders are interested in using molecular markers to assist with selection. Simple Sequence Repeat markers (SSRs) have many advantages, and SSRs developed from one species can sometimes be used with related species. Six SSRs derived from the weed R. alceifolius, and 74 SSRs from R. idaeus red raspberry `Glen Moy' were tested on R. idaeus red raspberry selection NY322 from Cornell Univ., R. occidentalis `Jewel' black raspberry, Rubus spp. blackberry `Arapaho', and blackberry selection APF-12 from the Univ. of Arkansas. The two raspberry genotypes are parents of an interspecific mapping population segregating for primocane fruiting and other traits. The two blackberry genotypes are parents of a population segregating for primocane fruiting and thornlessness. Of the six R. alceifolius SSRs, two amplified a product from all genotypes. Of the 74 red raspberry SSRs, 56 (74%) amplified a product from NY322, 39 (53%) amplified a product from `Jewel', and 24 (32%) amplified a product from blackberry. Of the 56 SSRs that amplified a product from NY322, 17 failed to amplify a product from `Jewel' and, therefore, detected polymorphisms between the parents of this mapping population. Twice as many detected polymorphisms of this type between blackberry and red raspberry, since 33 SSRs amplified a product from NY322, but neither of the blackberry genotypes. Differences in PCR product sizes from these genotypes reveal additional polymorphisms. Rubus is among the most diverse genera in the plant kingdom, so it is not surprising that only 19 of the 74 raspberry-derived SSRs amplified a product from all four of the genotypes tested. These SSRs will be useful in interspecific mapping and cultivar development.

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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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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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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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Eric T. Stafne, John R. Clark, Courtney A. Weber, Julie Graham, and Kim S. Lewers

1 To whom reprint requests should be addressed. E-mail: lewersk@ba.ars.usda.gov . The authors wish to thank Ms. Kate Rappaport for SSR testing reactions and Ms. Tina Sphon for reaction analyses. Thanks also to Dr. Tad Sonstagard for managing, and

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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.