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Kang Hee Cho, Seo Jun Park, Su Jin Kim, Se Hee Kim, Han Chan Lee, Mi Young Kim, and Jae An Chun

to distinguish the 45 blueberry cultivars according to the present or absent and size of PCR product bands. Table 3. Sequence-characterized amplified region (SCAR) primer pairs derived from cloned random amplified polymorphic DNA (RAPD) marker

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Kang Hee Cho, Jung Ho Noh, Seo Jun Park, Se Hee Kim, Dae-Hyun Kim, and Jae An Chun

amplified, which made it easy to identify the grapevine cultivars. Table 3. Sixteen sequence-characterized amplified region (SCAR) primer pairs derived from cloned randomly amplified polymorphic DNA (RAPD) markers sequences. A 334-bp fragment was amplified

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Hong Xu and Alan T. Bakalinsky

Five sequence characterized amplified region (SCAR) DNA markers were reevaluated at substantially higher annealing temperatures than originally reported; four were polymorphic among nine rootstocks tested. Four new informative SCAR markers also are reported, based on redesigning primers from previously cloned random amplified polymorphic DNA (RAPD) markers. Based on the eight polymorphic markers, rootstocks MG 420A, MG101-14, Richter 99, Couderc 3309, and Kober 5BB were distinguishable. Riparia Gloire and Couderc 1616 could be distinguished from the others, but not from one another, and SO4 and 5C also could be distinguished from the others, but not from one another.

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Jean-Guy Parent and Danièle Pagé

Five polymorphic random amplified polymorphic DNA (RAPD) markers for 13 red raspberry (Rubus idaeus L.) and two purple raspberry (R. idaeus L. × R. occidentalis L.) cultivars were cloned and their termini sequenced. Sequence-specific 24-mer primer pairs were synthesized as extended RAPD primers and used in sequence characterized amplified region (SCAR) DNA analysis. All primer pairs generated polymorphic SCAR markers of the original RAPD marker sizes and length variants. Markers from four of the primer pairs could be easily scored and were adequate to identify the raspberry cultivars of the certification program of the province of Quebec.

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Thomas Horejsi, Jodie M. Box, and Jack E. Staub

The conversion of randomly amplified polymorphic DNA (RAPD) markers to sequence characterized amplified region (SCAR) markers, and the effects of differing polymerase chain reaction (PCR) conditions were studied in cucumber (Cucumis sativus L.). Attempts were made to clone and sequence 75 RAPD PCR products to produce SCAR primers (16 to 22 nucleotides) designed to amplify original RAPD PCR products. The influence of template DNA source, purity, and concentration, MgCl2 concentration, Taq polymerase source, and type of thermocycler upon RAPD and SCAR marker performance was evaluated. Conversion of RAPD to SCAR markers was not universally successful, and SCAR primers reacted differently to varying PCR conditions. Only 48 (64%) of 75 RAPD markers were successfully converted to SCAR markers and 11 (15%) of these reproduced the polymorphism observed with the original RAPD PCR product. Moreover, some SCAR primer pairs produced multiple polymorphic PCR products. The band intensity of SCAR markers were brighter (P = 0.05) than their corresponding RAPD markers with only one exception. The SCAR markers examined were less influenced (P = 0.05) by MgCl2 concentration than their corresponding RAPD markers. However, some SCAR markers were more sensitive to reaction impurities than their RAPD counterparts and SCAR markers tended to be less readily visualized (decrease in frequency of visible PCR product) with low concentrations (1 and 2 mm) of template DNA than their corresponding RAPD markers. Neither the source of Taq nor the type of thermocycler used affected the performance of SCAR and RAPD markers. These data suggest that although SCAR markers may demonstrate enhanced performance over the RAPD markers from which they are derived, careful consideration must be given to both the costs and potential benefits of SCAR marker development in cucumber.

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Janel L. Giovannelli, Mark W. Farnham, Min Wang, and Allan E. Strand

Downy mildew, caused by the fungal parasite Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of Brassica oleracea L. crops, including broccoli (B. oleracea, Italica Group). The development and deployment of downy mildew resistant broccoli cultivars is a priority for breeders and producers. Identification of genetic markers linked to downy mildew resistance genes should facilitate selection for resistance and pyramiding of resistance genes into cultivars. The objectives of this study were to 1) identify RAPD markers linked to a single dominant gene for resistance in broccoli, 2) clone and sequence the linked RAPD markers, and 3) develop and evaluate SCAR markers as screening tools for resistance. Bulked segregant analysis led to the identification of eight linked RAPD markers following a screen of 848 decamers. Two of the linked RAPD fragments, UBC359620 and OPM16750, were converted to dominant SCAR markers linked in coupling to the resistance locus at 6.7 and 3.3 cM, respectively. The SCAR marker based on UBC359620 sequence exhibited less accuracy (94%) than the original RAPD (96%) in differentiating resistant and susceptible plants, but the accuracy (97%) of the OPM16750-SCAR was not different than the original RAPD. These SCAR markers are among the first genetic markers found linked to a gene conferring cotyledon-stage downy mildew resistance in B. oleracea. Results of this work provide breeders with useful information and tools for the systematic development of resistant cultivars.

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Soon O. Park, Kevin M. Crosby, Rongfeng Huang, and T. Erik Mirkov

Male sterility is an important trait of melon in F1 hybrid seed production. Molecular markers linked to a male-sterile gene would be useful in transferring male sterility into fertile melon cultivars and breeding lines. However, markers linked to the ms-3 gene for male sterility present in melon have not been reported. Our objectives were to identify randomly amplified polymorphic DNA (RAPD) markers linked to the ms-3 gene controlling male sterility using bulked segregant analysis in an F2 population from the melon cross of line ms-3 (male-sterile) × `TAM Dulce' (male-fertile), convert the most tightly linked RAPD marker to the ms-3 gene into a sequence characterized amplified region (SCAR) marker based on a specific forward and reverse 20-mer primer pair, and confirm the linkage of the RAPD and SCAR markers with the ms-3 gene in an F2 population from the cross of line ms-3 × `Mission' (male-fertile). A single recessive gene controlling male sterility was found in F2 individuals and confirmed in F3 families. Two RAPD markers that displayed an amplified DNA fragment in the male-sterile bulk were detected to be linked to the ms-3 gene in the F2 population from the cross of line ms-3 × `TAM Dulce'. RAPD marker OAM08.650 was closely linked to the ms-3 gene at 2.1 cM. SCAR marker SOAM08.644 was developed on the basis of the specific primer pair designed from the sequence of the RAPD marker OAM08.650. The linked RAPD and SCAR markers were confirmed in the F2 population from the cross of line ms-3 × `Mission' to be consistently linked to the ms-3 gene at 5.2 cM. These markers were also present in 22 heterozygous fertile F1 plants having the ms-3 gene. The RAPD and SCAR markers linked to the ms-3 gene identified, and confirmed here could be utilized for backcrossing of male sterility into elite melon cultivars and lines for use as parents for F1 hybrid seed production.

Open access

Melinda A. Miller-Butler, Barbara J. Smith, Brian R. Kreiser, and Eugene K. Blythe

rating following inoculation with three species of Colletotrichum , and presence or absence of sequence characterized amplified region (SCAR) markers and alleles in this study and in a European study ( Lerceteau-Köhler et al., 2005 ). During

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Karen R. Harris, Kai-Shu Ling, W. Patrick Wechter, and Amnon Levi

Coulter CEQ8800 DNA Genetic Analysis System. Generation of a ZYRP-SCAR marker for identifying polymorphism using capillary electrophoresis. A sequence-characterized amplification region (SCAR) marker was generated based on the single nucleotide

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Xuelin Shen, Yanmei Zhang, Zhao Lei, Yibo Lin, Minxu Cao, Yueyu Hang, and Xiaoqin Sun

-specific fragment amplified using primer intersimple sequence repeat-880. The underlined sequences were designed sequence-characterized amplified region primers. A pair of SCAR primers was designed as SCAR-F and SCAR-R (underlined in Fig. 4 ), corresponding to the