, alternative methods have been developed to detect B. aclada in onion bulbs. These include culturing samples on semiselective media, enzyme-linked immunosorbent assay tests, and conventional polymerase chain reaction (PCR) detection ( Kritzman and Netzer
Timothy W. Coolong, Ronald R. Walcott, and William M. Randle
Chu-Hui Chiang, Tsong-Ann Yu, Shu-Fang Lo, Chao-Lin Kuo, Wen-Huang Peng, and Hsin-Sheng Tsay
was stored at –20 °C. Table 1. List of 20 Dendrobium species with their voucher and GenBank accession numbers for multiplex polymerase chain reaction and amplification refractory mutation system analyses. Polymerase chain reaction amplification and
Yan Hong and SiLan Dai
function, primer sequences, amplicon length, optimum annealing temperature (Tm), polymerase chain reaction efficiency (PE), regression coefficient ( R 2 ), and quantification cycle (Cq) value. z Primer design and test of amplification efficiency. Primers
Xinwang Zhang, Ikuo Nakamura, and Masahiro Mii
), DNA amplification fingerprinting ( Cerny et al., 1996 ), and RFLP analysis of chloroplast DNA ( Ando et al., 2005a ). As an alternative, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis, which is simple, rapid
Tao Wang, Ruijie Hao, Huitang Pan, Tangren Cheng, and Qixiang Zhang
-level comparisons among Prunus species. We believe the availability of the mei genome will stimulate research in the functional genomics of the Prunus genus. Real-time reverse transcription polymerase chain reaction is a sensitive method that is widely used to
Allan F. Brown, Elizabeth H. Jeffery, and John A. Juvik
in a population created by crossing broccoli and cabbage, only 2% of the F 2 plants produced cabbage-like heads and none of these were of commercial grade. The development of a linkage map specific to broccoli using polymerase chain reaction (PCR
Amnon Levi, William P. Wechter, Karen R. Harris, Angela R. Davis, and Zhangjun Fei
et al., 2001 , 2006b , 2009 ), which may not be readily detected by random primers. In fact, DNA polymorphism among watermelon cultivars could be detected with expressed sequence tag (EST)-polymerase chain reaction (PCR) and sequence
B. Sosinski and D.S. Douches
DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.
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.
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.