Abstract
A simple, PCR-based method has been developed for the rapid genoty** of large numbers of samples. The method involves a alkaline extraction of DNA from plant tissue using a slight modification of the procedure of Wang et al. (Nucleic Acids Res 21:4153–4154, 1993). Template DNA is amplified using allelespecific associated primers (ASAPs) which, at stringent annealing temperatures, generate only a single DNA fragment and only in those individuals possessing the appropriate allele. This approach eliminates the need to separate amplified DNA fragments by electrophoresis. Instead, samples processing the appropriate allele are identified by direct staining of DNA with ethidium bromide. Total technician time required for extraction, amplification and detection of 96 samples is about 4 h, and this time requirement can be reduced by automation. Excluding labor, cost per sample is less than $0.40. The method is tested using the codominant isozyme marker, alcohol dehydrogenase (Adh-1) gene in pea (Pisum sativum), and applied to the screening of photoperiod genes in common bean (Phaseolus vulgaris L.).
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Chunwongse J, Martin GB, Tanksley SD (1993) Pre-germination genotypic screening using PCR amplification of half-seeds. Theor Appl Genet 86:694–698
Gu W-K, Weeden NF, Wallace DH (1993) A DNA marker for ppd, a gene conferring insensitivity to photoperiod in common bean. Annu Rep Bean Improv Coop 36:1–2
Gu W-K, Weeden NF, Zhu J-Q, Wallace DH (1994) Identification of a DNA marker for Hr, a gene that interacts with Ppd to confer extreme photoperiod sensitivity in common bean. Annu Rep Bean Improv Coop 37:125–126
Heller DP, Greenstock CL (1994) Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye. Biophys Chem. 50:305–312
Kesseli RV, Paran I, Michelmore RW (1992) Efficient map** of specifically targeted genomic regions and the tagging of these regions with reliable PCR-based genetic markers. In: Applications of RAPD technology to plant breeding (Joint plant breeding symposia series). ASSA ASHS AGA, Minneapolis, Minn., pp 31–35
Klimyuk VI, Carroll BJ, Thomas CM, Jones JDG (1993) Alkali treatment for rapid preparation of plant material for reliable PCR analysis. Plant J 3:493–494
Paran I, Michelmere RW (1992) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theor Appl Genet 85:985–993
Ragot M, Hoisington DA (1993) Molecular markers for plant breeding: comparison of RFLP and RAPD genoty** costs. Theor Appl Genet 86:975–984
Timmerman GM, Frew TJ, Weeden NF, Miller AL, Goulden DS (1994) Linkage map** of er, a recessive Pisum sativum gene for resistance to powdery mildew fungus (Erysiphe pisi) Theor Appl Genet 88:1050–1055
Torres A, Weeden NF, Martin A (1993) Linkage among isozyme, RFLP and RAPD markers in Vicia faba. Theor Appl Genet 85:937–945
Wallace DH, Yourstone KS, Masaya PN, Zobel RW (1993) Photo-period gene control over partitioning between reproductive vs. vegetative growth. Theor Appl Genet 86:6–16
Wang H, Qi M, Adrian JC (1993) A simple method of preparing plant samples for PCR. Nuclei Acids Res 21:4153–4154
Weeden NF, Provvidenti R (1988) A marker locus, Adh-1, for resistance to Pea Enation Mosaic Virus in Pisum sativum. J Hered 79:128–131
Weeden NF, Muehlbauer FJ, Ladizinsky G (1992) Extensive convervation of linkage relationships between pea and lentil genetic maps. J Hered 83:123–129
Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18:7213–7218
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535
Xu L, Hall BG (1994) SASA: a simplified, reliable method for allele-specific amplification of polymorphic sites. Bio Techniques 16:171–172
Yu J, Gu WK, Provvidenti R, Weeden NF (1995) Identification and map** of two DNA markers linked to the gene conferring resistance to pea enation mosaic virus. J Amer Soc Hort Sci (in press)
Zubay GL (1988) Biochemistry. Macmillan, New York
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Communicated by J. S. Beckmann
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Gu, W.K., Weeden, N.F., Yu, J. et al. Large-scale, cost-effective screening of PCR products in marker-assisted selection applications. Theoret. Appl. Genetics 91, 465–470 (1995). https://doi.org/10.1007/BF00222974
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DOI: https://doi.org/10.1007/BF00222974