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Nobuhito Mitani, Atsushi Kono, Masahiko Yamada, Akihiko Sato, Shozo Kobayashi, Yusuke Ban, Toshihito Ueno, Mikio Shiraishi, Shinya Kanzaki, Tomoyuki Tsujimoto, and Keizo Yonemori

. Lane 9: offspring presumed to be non-PCNA displaying the A 1 . Lanes 2, 5, 8: offspring presumed to be non-PCNA displaying A 3 . Lanes 6: offspring presumed to be PCNA. M: molecular marker (50 bp ladder; Roche Diagnostics, Tokyo, Japan). SCAR = sequence-characterized

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Juan M. Osorno, Carlos G. Muñoz, James S. Beaver, Feiko H. Ferwerda, Mark J. Bassett, Phil N. Miklas, Teresa Olczyk, and Bill Bussey

-9 with resistance derived from G35172 had the susceptible band (530 bp) from the SR-2 sequence-characterized amplified region (SCAR) marker based on a codominant randomly amplified polymorphic DNA diagnostic for presence or absence of the bgm gene for

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Hong Xu, Diane J. Wilson, S. Arulsekar, and Alan T. Bakalinsky

Randomly amplified polymorphic DNA (RAPD) markers were generated for identifying grape (Vitis) rootstocks. Seventy-seven primers (10 bases long) were screened using CsCl-purified leaf DNA derived from several field samples of nine rootstocks sampled in successive years. Nine RAPD markers were detected from six primers and, in combination, distinguished all nine rootstocks tested. Because inconsistencies were encountered in performing the RAPD assay, sequence-specific primers were derived from cloned RAPD bands for use under more stringent amplification conditions. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In some cases, bands of the same size generated by the same primer in different rootstocks-normally scored as the same marker-failed to cross-hybridize, implying lack of homology between the bands. More commonly, bands scored as absent based on ethidium bromide staining were detected by hybridization. Six of the nine cloned RAPD bands were partially sequenced, and sequence-specific primer pairs were synthesized. Two primer pairs amplified a product the same size as the original RAPD band in all rootstocks, resulting in loss of polymorphism. Two other pairs of sequence-specific primers derived from the same marker failed to amplify the expected band consistently. Three of the most useful primer pairs amplified apparent length variants in some accessions and will have value as polymerase chain-reaction markers for fingerprinting.

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Yanmei Zhang, Xuelin Shen, Xiaoqin Sun, Jia Liu, Yifeng Xia, Xin Zou, and Yueyu Hang

’-specific fragment amplified using primer RAPD26. The underlined sequences are designed sequence characterized amplified region primers. Fig. 3. Verification of the ‘Heshangling’-specific randomly amplified polymorphic DNA–sequence characterized amplified region by

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Patrick D. O'Boyle, James D. Kelly, and William W. Kirk

; Singh and Munoz, 1999 ). The P. acutifolius -derived QTL examined in this research are two independent QTL linked to the sequence-characterized amplified region (SCAR) markers BC420 and SU91 that map to bean linkage groups B6 and B8, respectively

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Jean-Guy Parent and Daniele Page

Random amplified polymorphic DNA (RAPD) markers are used in Quebec's certification program to verify the identity of raspberry cultivars. However, sequence characterized amplified region (SCAR) markers, less sensitive to modifications in reaction conditions, could be derived from RAPD markers. Our objective was to evaluate the potential of SCAR markers to replace the RAPD ones. Five RAPD markers obtained with primer OPG06 (length of 520, 700, 825, 1450, and 2000 bp) were cloned in pTZ/PC or pCRII vectors. Extremities of the cloned markers were sequenced by the nonradioactive silver sequence method using pUC/M13 forward and reverse primers. Sequence information was used to make SCAR primers, similar in length to standard PCR primers. Some SCAR primers were elongated RAPD primers, whereas others were from internal regions. Ability of primer pairs and combination of primer pairs to discriminate cultivars of our certification program was compared with their RAPD counterparts as well as with the technical feasibility of both methods.

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Renbing Zhang, Yong Xu, Ke Yi, Haiying Zhang, Ligong Liu, Guoyi Gong, and Amnon Levi

A genetic linkage map was constructed for watermelon using 117 recombinant inbred lines (RILs) (F2S7) descended from a cross between the high quality inbred line 97103 [Citrullus lanatus var. lanatus (Thunb.) Matsum. & Nakai] and the Fusarium wilt (races 0, 1, and 2) resistant U.S. Plant Introduction (PI) 296341 (C. lanatus var. citroides). The linkage map contains 87 randomly amplified polymorphic DNA (RAPD) markers, 13 inter simple sequence repeat (ISSR) markers, and four sequenced characterized amplified region (SCAR) markers. The map consists of 15 linkage groups. Among them are a large linkage group of 31 markers covering a mapping distance of 277.5 cM, six groups each with 4 to 12 markers covering a mapping distance of 51.7 to 172.2 cM, and eight small groups each with 2-5 markers covering a mapping distance of 7.9 to 46.4 cM. The map covers a total distance of 1027.5 cM with an average distance of 11.7 cM between two markers. The map is useful for the further development of quantitative trait loci (QTLs) affecting fruit qualities and for identification of genes conferring resistance to Fusarium wilt (races 0, 1 and 2). The present map can be used for further construction of a reference linkage map for watermelon based on an immortalized mapping population with progenies homozygous for most gene loci.

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Zhan'ao Deng, Fred G. Gmitter Jr., Shunyuan Xiao, and Shu Huang

Citrus tristiza virus (CTV) is the most-significant viral pathogen of citrus in the world. Rapid decline of trees on sour orange and stem pitting of grapefruit and sweet orange, two diseases induced by CTV, severely jeopardize citrus production worldwide. It is recognized that all future rootstocks should be resistant to this virus, and scion resistance to stem pitting stains is desirable. To facilitate introgression of the CTV resistance gene from Poncirus trifoliata and development of CTV-resistant varieties in citrus, gene mapping projects have been initiated and more than a dozen RAPD markers have been identified with tight linkage to the resistance gene. As part of our efforts to use marker-assisted selection with a large number of crosses, and ultimately to accomplish map-based cloning of the CTV resistance gene, we have been converting the most tightly linked RAPD markers into SCAR (sequence characterized amplified region) markers by cloning, sequencing the marker fragments, and designing locus-specific primers. One codominant and several dominant SCARs have been developed thus far. The updated progress and utilization of these SCARs in marker-assisted selection and possibly in characterization of a BAC library will be presented and discussed.

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Amnon Levi, Claude E. Thomas, Xingping Zhang, Tarek Joobeur, Ralph A. Dean, Todd C. Wehner, and Bruce R. Carle

A genetic linkage [randomly amplified polymorphic DNA (RAPD)-based] map was constructed for watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai] using a BC1 population [PI 296341-fusarium wilt resistant × New Hampshire Midget (fusarium susceptible)] × `New Hampshire Midget'. The map contains 155 RAPD markers, and a 700-base pair sequenced characterized amplified region (SCAR) marker that corresponds to a fragment produced by the RAPD primer GTAGCACTCC. This marker was reported previously as linked (1.6 cM) to race 1 fusarium wilt resistance in watermelon. The markers segregated to 17 linkage groups. Of these, 10 groups included nine to 19 markers, and seven groups included two to four markers. The map covers a genetic linkage distance of 1295 cM. Nine of the 10 large linkage groups contained segments with low (or no) level of recombination (0 to 2.6 cM) among markers, indicating that the watermelon genome may contain large chromosomal regions that are deficient in recombination events. The map should be useful for identification of markers linked closely to genes that control fruit quality and fusarium wilt (races 1 and 2) resistance in watermelon.

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K.M. Haymes, W.E. Van de Weg, P. Arens, J.L. Maas, B. Vosman, and A.P.M. Den Nijs

Two dominant sequence characterized amplified region (SCAR) markers (linked at 3.0 cM, coupling phase) were constructed for the strawberry (Fragaria ×ananassa Duch.) gene Rpf1. This gene confers resistance to red stele root rot, caused by the soil-born fungus Phytophthora fragariae Hickman var. fragariae. The SCAR markers were developed originally from the sequence of RAPD OPO-16C(438) that is linked in repulsion phase to the Rpf1 allele. This SCAR primer set produced multiple bands in the resistant test progeny and in some of the susceptible progeny; therefore, new SCARs were developed based on the sequence differences among these bands. These new SCARs were linked in coupling phase to the Rpf allele and mapped to the same location as the original RAPD OPO-16C(438). The SCAR markers, as well as some additional RAPD markers known to be linked to Rpf1, were shown to be highly conserved in linkage to the gene based on examination of 133 European and North American Fragaria L. sp. cultivars and breeding selections. These flanking RAPD and SCAR-PCR markers can be used in breeding programs for the selection of red stele (Rpf1) resistance.