Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques |
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Authors: | Jhy-Jhu Lin Jonathan Kuo Jin Ma James A Saunders Hunter S Beard Margaret H MacDonald William Kenworthy George N Ude Benjamin F Matthews |
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Institution: | (1) Life Technologies, Inc., 8717 Grovemont Circle, 20877 Gaithersburg, MD, USA;(2) Climate Stress Laboratory, USDA ARS, 20705 Beltsville, MD, USA;(3) Plant Molecular Biology Laboratory, USDA ARS, 20705 Beltsville, MD, USA;(4) Department of Agronomy, University of Maryland, 20742 College Park, MD, USA |
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Abstract: | Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability
to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars
were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic
bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease
restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished
on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained
with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in
those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands
using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of
the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish
polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP
is the most useful. |
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Keywords: | DNA fingerprinting Glycine max PCR |
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