SUBTRACTIVE HYBRIDIZATION AS A TOOL TO SCREEN FOR GENES INVOLVED IN DORMANCY AND GROWTH IN UNDERGROUND ADVENTITIOUS BUDS OF LEAFY SPURGE (EUPHORBIA ESULA)

Authors: JIA, YING - NDSU; Gu, Yong; Horvath, David; Anderson, James; Chao, Wun

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/7/2005
Publication Date: 2/7/2005
Citation: Jia, Y., Gu, Y., Horvath, D.P., Anderson, J.V., Chao, W.S. 2005. Subtractive hybridization as a tool to screen for genes involved in dormancy and growth in underground adventitious buds of leafy spurge (Euphorbia esula). [Abstract]. Weed Science Society of America. Page No. 120.

Interpretive Summary: Leafy spurge is a noxious perennial weed that infests range and recreational lands in the Northern Great Plains. Underground adventitious buds (crown and root buds) of leafy spurge are a key component responsible for its proliferation and persistence as a perennial weed. These buds develop during the normal growing season but are maintained in a quiescent state through correlative inhibition (or paradormancy). In the fall, these buds develop innate dormancy prior to over wintering. To gain a better understanding of bud dormancy and growth in leafy spurge, we developed two subtracted cDNA libraries to search for differentially-expressed genes in dormant or growing buds. To identify individual clones represented in the libraries, 15,744 clones from both libraries were spotted onto membranes and screened several times by hybridizion with radioactive probes generated from different pools of cDNA clones known to be redundant. Eventually, 2,304 non-hybridizing clones were sequenced and found to represent 515 individual clones. DNA inserts from the 515 individual clones were PCR-amplified, gel-purified, and spotted onto Hybond-N+ membrane for macroarray analysis. Radioactive probes were made from RNAs extracted from crown buds of intact (control) and a series of induced (2h, 2d, and 4d after decapitation) plants. Macroarray analyses identified 66 differentially-expressed clones. Semi quantitative RT-PCR was used to confirm macroarray results. RT-PCR was also used to monitor expression of other transcripts that were not sufficiently expressed in the buds to allow detection by hybridization. Currently, 14 up- or down- regulated cDNA clones have been confirmed by RT-PCR analyses.

Technical Abstract: Leafy spurge is a noxious perennial weed that infests range and recreational lands in the Northern Great Plains. Underground adventitious buds (crown and root buds) of leafy spurge are a key component responsible for its proliferation and persistence as a perennial weed. These buds develop during the normal growing season but are maintained in a quiescent state through correlative inhibition (or paradormancy). In the fall, these buds develop innate dormancy prior to over wintering. To gain a better understanding of bud dormancy and growth in leafy spurge, we developed two subtracted cDNA libraries (forward and reverse). The forward library contains copies of genes preferentially-expressed in growing root buds, whereas the reverse library contains genes preferentially-expressed in dormant buds. Random sequencing of 100 cDNA clones from each cDNA library revealed that these libraries contained many highly redundant clones. To identify individual clones represented in the libraries, 15,744 clones from both libraries were spotted onto membranes and screened several times by hybridizion with radioactive probes generated from different pools of cDNA clones known to be redundant. Eventually, 2,304 non-hybridizing clones were sequenced and found to represent 515 individual clones. DNA inserts from the 515 individual clones were PCR-amplified, gel-purified, and spotted onto Hybond-N+ membrane for macroarray analysis. Radioactive probes were made from RNAs extracted from crown buds of intact (control) and a series of induced (2h, 2d, and 4d after decapitation) plants. Macroarray analyses identified 66 differentially-expressed clones. Semi quantitative RT-PCR was used to confirm macroarray results. RT-PCR was also used to monitor expression of other transcripts that were not sufficiently expressed in the buds to allow detection by hybridization. Currently, 14 up- or down- regulated cDNA clones have been confirmed by RT-PCR analyses. Some of these genes play important roles in cell metabolism such as glutamate metabolism and pantothenate (vitamin B5) and myo-inositol biosynthesis, while others are hormone-regulated or currently have unknown functions. Key words: subtractive hybridization, dormancy, leafy spurge, macroarray.