Roots of Centaurea maculosa grown in competition with Festcua idahoensis This is an experimental replicate of chip 720 (2-23-06)
Biomaterial provider
Callaway, University of Montana, Missoula
Treatment protocol
Centaurea maculosa grown in competition with Festuca idahoensis
Growth protocol
In April 2005, 1.5 liters of sterile 20/30 grit sand (Lane Mountain, Inc. Valley, WA) was added to the bottom of 2.4 liter pots and 1 liter of weed-free sifted soil collected in Missoula, MT was placed on top of the sand. The pots were placed in a naturally lit greenhouse supplemented with artificial light and seeded with five seeds of Gaillardia aristata (strong competitor) or Festuca idahoensis (weak competitor), species which are both native to North American rangelands invaded by Centaurea. Once seedlings were established they were thinned to one plant per pot. After the natives had grown for 30 days, a single Centaurea seed collected from an invasive population in Missoula, MT was added to each pot containing native species and allowed to establish. Centaurea seeds were also grown in pots without natives. Plants were fertilized every three weeks with 100 mL of Miracle GrowTM solution per pot (0.34 grams 15-2-20 Miracle GrowTM per liter water), watered to near saturation, and rotated on the bench every 10 days. Plants were harvested in September of 2005, when native plants were approximately 153 days old and C. maculosa plants were 123 days old
In April 2005, 1.5 liters of sterile 20/30 grit sand (Lane Mountain, Inc. Valley, WA) was added to the bottom of 2.4 liter pots and 1 liter of weed-free sifted soil collected in Missoula, MT was placed on top of the sand. The pots were placed in a naturally lit greenhouse supplemented with artificial light and seeded with five seeds of Gaillardia aristata (strong competitor) or Festuca idahoensis (weak competitor), species which are both native to North American rangelands invaded by Centaurea. Once seedlings were established they were thinned to one plant per pot. After the natives had grown for 30 days, a single Centaurea seed collected from an invasive population in Missoula, MT was added to each pot containing native species and allowed to establish. Centaurea seeds were also grown in pots without natives. Plants were fertilized every three weeks with 100 mL of Miracle GrowTM solution per pot (0.34 grams 15-2-20 Miracle GrowTM per liter water), watered to near saturation, and rotated on the bench every 10 days. Plants were harvested in September of 2005, when native plants were approximately 153 days old and C. maculosa plants were 123 days old
Extracted molecule
total RNA
Extraction protocol
RNeasy kit (quiagen)
Label
CY3
Label protocol
Genisphere Array900
Hybridization protocol
Genisphere Array900
Scan protocol
Axon GenePix3.0
Description
OAR27K 723, 3/17/06
Data processing
GenePix Pro (gpr) and image files for each experiment were uploaded to a secure site through the Yale server where they could be accessed for analysis (http://ymd.med.yale.edu/ymd_prod/cgi-bin/gz_login.cgi). In the preliminary experiment, all unflagged genes were considered in the analysis without analyzing up or down regulation between tissues, to get a general overview of hybridization. For each chip, flagged genes were removed from the analysis. The average background was calculated for each dye channel (wavelength 635 and 532) and only genes having pixel counts (median minus background) over two times the average background in at least one channel were considered. In Experiment 2 each chip was initially analyzed separately. Flagged values and all channel values that were less than two standard deviations above the mean background were removed from the analysis. Ratios ((F635-B635)/(F532-B532)) of the remaining values were averaged per chip, and a correction factor was calculated for each chip, in order to set the average of all ratios equal to one. Ratios were multiplied by the appropriate correction factor to globally normalize the data. We chose oligos (array spots) that were present in all four chips for further analysis. This raises our confidence in the data, and allows us to compare the two experimental conditions. In addition, normalization provided better agreement between results from the microarray and those obtained by RT-PCR. Replicates (two per experimental condition) were combined and the average ratios and respective standard deviations were calculated for each oligo. A one sample t-test was preformed to identify oligos in which the test condition (Centaurea-Gaillardia or Centaurea-Festuca) was significantly different from the control (Centaurea alone). A two sample t-test was preformed to identify oligos that were differentially regulated between experiments (Centaurea-Gaillardia and Centaurea-Festuca). Accession numbers for each gene fitting the above criteria were used to query the TAIR (www.arabidopsis.org) database to determine function, and genes were then grouped into functional categories based on the Kyoto Encyclopedia of Genes and Genomics (KEGG) Orthology System (www.genome.jp/kegg/brite.html).
