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Beibei Li, Jianfu Jiang, Xiucai Fan, Ying Zhang, Haisheng Sun, Guohai Zhang, and Chonghuai Liu

various DNA-based molecular markers, simple sequence repeats (SSRs), with the advantages of codominance inheritance, a high degree of polymorphism, reproducibility, and simple operation ( Sefc et al., 2001 ), have been widely used in genetic diversity

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Marie Pairon, Anne-Laure Jacquemart, and Daniel Potter

(SSR) markers covering the Prunus genome Theor. Appl. Genet. 106 819 825 Becher, S.A. Steinmetz, K. Weising, K. Boury, S. Peltier, D. Renou, J.-P. Kahl, G. Wolff, K

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Gennaro Fazio, Jack E. Staub, and Sang Min Chung

Highly polymorphic microsatellites or simple sequence repeat (SSR), along with sequence characterized amplified region (SCAR) and single nucleotide polymorphisms (SNP), markers are reliable, cost-effective, and amenable for large scale analyses. Molecular polymorhisms are relatively rare in cucumber (Cucumis sativus L.) (3% to 8%). Therefore, experiments were designed to develop SSR, SCAR and SNP markers, and optimize reaction conditions for PCR. A set of 110 SSR markers was constructed using a unique, strategically applied methodology that included the GeneTrapper (Life Technologies, Gaithersburg, Md.) kit to select plasmids harboring microsatellites. Of these markers, 58 (52%) contained dinucleotide repeats (CT, CA, TA), 21 (19%) possessed trinucleotide repeats (CTT, ATT, ACC, GCA), 3 (2.7%) contained tetranucleotide repeats (TGCG, TTAA, TAAA), 4 (3.6%) enclosed pentanucleotide repeat (ATTTT, GTTTT, GGGTC, AGCCC), 3 (2.7%) contained hexanucleotide repeats (CCCAAA, TAAAAA, GCTGGC) and 21 possessed composite repeats. Four SCARs (L18-3 SCAR, AT1-2 SCAR, N6-A SCAR, and N6-B SCAR) and two PCR markers based on SNPs (L18-2H19 A and B) that are tightly linked to multiple lateral branching (i.e., a yield component) were also developed. The SNP markers were developed from otherwise monomorphic SCAR markers, producing genetically variable amplicons. The markers L18-3 SCAR and AT1-2 SCAR were codominant. A three-primer strategy was devised to develop a codominant SCAR from a sequence containing a transposable element, and a new codominant SCAR product was detected by annealing temperature gradient (ATG) PCR. The use of a marker among laboratories can be enhanced by methodological optimization of the PCR. The utility of the primers developed was optimized by ATG-PCR to increase reliability and facilitate technology transfer. This array of markers substantially increases the pool of genetic markers available for genetic investigation in Cucumis.

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J. Steven Brown, R.J. Schnell, J.C. Motamayor, Uilson Lopes, David N. Kuhn, and James W. Borrone

A genetic linkage map was created from 146 cacao trees (Theobroma cacao), using an F2 population produced by selfing an F1 progeny of the cross Sca6 and ICS1. Simple sequence repeat (SSR) markers (170) were used principally for this map, with 12 candidate genes [eight resistance gene homologues (RGH) and four stress related WRKY genes], for a total of 182 markers. Joinmap software was used to create the map, and 10 linkage groups were clearly obtained, corresponding to the 10 known chromosomes of cacao. Our map encompassed 671.9 cM, approximately 100 cM less than most previously reported cacao maps, and 213.5 cM less than the one reported high-density map. Approximately 27% of the markers showed significant segregation distortion, mapping together in six genomic areas, four of which also showed distortion in other cacao maps. Two quantitative trait loci (QTL) for resistance to witches' broom disease were found, one producing a major effect and one a minor effect, both showing important dominance effects. One QTL for trunk diameter was found at a point 10.2 cM away from the stronger resistance gene. One RGH flanked the minor QTL for witches' broom resistance, implying possible association. QTLs mapped in F2 populations produce estimates of additive and dominance effects, not obtainable in F1 crosses. As dominance was clearly shown in the QTL found in this study, this population merits further study for evaluation of dominance effects for other traits. This F2 cacao population constitutes a useful link for genomic studies between cacao and cotton, its only widely grown agronomic relative.

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Hong Lin and M. Andrew Walker

probe and primers used in RFLP and SSR analysis and Doug Adams and Sekar Arulsekar for critiquing this manuscript. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must

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Luisa Monte-Corvo, Luis Goulão, and Cristina Oliveira

Inter-simple sequence repeat (ISSR) markers were used for cultivar identification and for determination of the phenetic relationships among 24 pear cultivars (Pyrus communis L.). The ability of several molecular marker systems including randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), inter-simple sequence repeats (ISSR), simple sequence repeats (SSR), and selective amplification of microsatellite polymorphic loci (SAMPL) to detect variation among clones of the most significant Portuguese cultivar, Rocha, was also investigated. Each of the eight ISSR primers tested was able to distinguish the 24 pear cultivars. The ISSR primers generated 337 markers, 79.5% of which were polymorphic. The cultivar dendrogram obtained with the ISSR marker data was very similar to that obtained with previous RAPD+AFLP analysis, confirming the genetic divergence of `Pérola', `Carvalhal' and `Lawson' from the other cultivars. Eight out of 15 apple [Malus sylvestris (L.) Mill. var domestica (Borkh.) Mansf.] SSR primers tested also amplified microsatellites in pear. None of the five molecular marker systems analyzed (with a total of 1082 markers) detected reproducible polymorphisms among the nine `Rocha' clones, in spite of the presence of clear phenotypic differences.

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Margaret R. Pooler, Louise G.H. Riedel, S.E. Bentz, and A.M. Townsend

Controlled pollinations were made between five hemlock (Tsuga) species from eastern North America and Asia, resulting in over 5700 germinating seedlings. A subset of putative hybrid seedlings from each cross was tested for authenticity by various DNA marker systems. The most reliable and useful system for verifying hybrids was amplified fragment-length polymorphism (AFLP) markers. Hybridizations between the eastern North American species, T. canadensis [L.] Carriere and T. caroliniana Engelm., and the Asian species, T. chinensis (Franch.) E. Pritz., were used as a model to test the inheritance, reliability, and ease of use of these markers. Using AFLP markers, we were able to verify 58 hybrids between T. caroliniana and T. chinensis, one hybrid between T. caroliniana and T. canadensis, but could find no definitive hybrids between T. canadensis and T. chinensis. Results using other marker systems, including RAPD, SCAR, ITS, and SSR, are also presented.

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Yayeh Zewdie, Michael J. Havey, James P. Prince, and Maria M. Jenderek

thank Drs. John McCallum (Crop and Food Research, Lincoln, New Zealand) and Jernej Jakse (Univ. of Wisconsin) for SSR primer sequences. We also thank Linda Donnelly (California State Univ., Fresno) for her technical assistance. Names are necessary to

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Anne Frary, Hasan Özgür Şığva, Ayfer Tan, Tuncer Taşkın, Abdullah İnal, Sevgi Mutlu, Mehmet Haytaoğlu, and Sami Doğanlar

al., 2007 ; Staub et al., 2004 ; Yildiz et al., 2011 )], simple sequence repeats [SSRs ( Danin-Poleg et al., 2001 ; Monforte et al., 2003 )], intersimple sequence repeats [ISSRs ( Perl-Treves et al., 1998 ; Yildiz et al., 2011 )], AFLPs

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Samuel G. Obae, Mark H. Brand, Bryan A. Connolly, Rochelle R. Beasley, and Stacey L. Lance

sequence repeat (SSR) markers. SSR markers are hypervariable, codominantly inherited, and widely distributed in the genome making them very informative for plant studies ( Kalia et al., 2011 ). Furthermore, SSR markers are sometimes transferable to closely