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United States Department of Agriculture

Agricultural Research Service

Title: A Combined Genomic Subtractive Hybridization and Bulked Segeregant Analysistechnique to Isolate DNA Markers Closely Linked to the Rps1-K Gene in Soybean.

item Vantoai, Tara
item Lohnes, David - OHIO STATE UNIVERSITY
item Specht, James - UNIV OF NE
item St Martin, Steven - OHIO STATE UNIVERSITY

Submitted to: Plant and Animal Genome
Publication Type: Abstract Only
Publication Acceptance Date: January 18, 1998
Publication Date: N/A

Technical Abstract: In order to identify molecular markers linked to Rps1-K, a dominant gene conferring resistance to Phytophthora sojae in soybean, we combined two different techniques(1) the Bulked Segregant Analysis, a method which facilitates the isolation of markers that are tightly linked to the locus of interest, and (2) DNA subtractive hybridization, a technique for directly identifying differences between two DNA samples referred to as tester and driver. Genotypes of F2 individuals of a population segregating at Rps1 locus were determined by the phenotypes of F23 progeny. The homozygous resistant F2 individuals were bulked to create a resistant DNA sample. Similarly a susceptible DNA sample was created by bulking the homozygous susceptible F2 individuals. The DNA samples were digested with EcoR1, separated by agarose gel electrophoresis, and divided into small, medium and big size fragments samples by gel slicing. The small fragment samples were directly ligated to EcoR1 adaptors and amplified by PCR. The medium and big fragments were digested with MseI first, then ligated with MseI adaptors and amplified by PCR. These PCR products were referred to as amplicons. DNA subtractive hybridizations were carried out by using resistant amplicons as testers and the corresponding susceptible amplicons as drivers. Different DNA subtractive hybridization procedures were compared. Four, one, and six sharp bands were identified from small, medium and big fragment groups respectively after three to five rounds of subtractive hybridization. The results were consistent in two independent DNA Subtractive Hybridization experiments. These bands were cloned and proved present only in the resistant parental genomic DNA by Southern analysis.

Last Modified: 4/22/2015
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