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United States Department of Agriculture

Agricultural Research Service

Title: Membrane Array and Rt-Pcr Analysis of Two Subtraction Cdna Libraries for Nematode Infected Soybean Roots

Authors
item Tucker, Mark
item Raina, Anjana
item Thai, Vanessa - GEORGE MASON UNIVERSTIY
item Xue, Ping - UNIVERSITY OF MARYLAND

Submitted to: Society of Nematologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: November 22, 2003
Publication Date: November 22, 2003
Citation: Tucker, M.L., Raina, A., Thai, V., Xue, P. 2004. Membrane array and rt-pcr analysis of two subtraction cdna libraries for nematode infected soybean roots. Society of Nematologists Proceedings. Abstract 149.

Technical Abstract: Soybean cyst nematode (SCN) is the most economically destructive pathogen of soybeans. Identification of gene expression that is upregulated during infection by SCN and also highly specific to the infection site will provide additional tools to engineer SCN resistance in soybean. We have prepared two subtraction libraries to enrich for SCN-induced genes expressed in the early and late stages of the 30-day lifecycle of SCN in soybean roots. In addition to subtraction with cDNA prepared from uninoculated roots, cDNA from SCN eggs were also used for subtraction. Two thousand cDNA clones were selected from each library and stored in 96-well microtiter plates. From each of these libraries 384 cDNAs were sequenced from both directions and the cDNAs arrayed onto nitrocellulose membranes. The membrane arrays were then hybridized to radioactively labeled cDNAs prepared from total root RNA isolated at 4, 12, and 20 days post-SCN-inoculation (PSI) and RNA from 0, 4, 12 and 20 days incubation with no inoculation. The 384 cDNAs from the early subtraction library (1, 2 and 4 days PSI) do not include any SCN transcripts and no host genes that were significantly upregulated at our conditions. However, 190 of the 384 cDNAs from the late subtraction library (8, 12, 20 days PSI) have high sequence identity with nematode sequences and most were associated with GenBank SCN ESTs from maturing nematodes. Moreover, a large number of the soybean cDNAs that were upregulated by the SCN infection in the late library were associated with previously identified nodulation (nod) genes. In addition to nod genes, there were a few upregulated cDNAs that were not previously reported to be associated with SCN infection and have been examined further by RT-PCR.

Last Modified: 7/28/2014
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