6 protocols
NimbleGen One-Color Array Scanning (Expression)
NimbleGen Hybridization & Washing (Expression)
NimbleGen Sample Labeling (Expression)
Following stratification procedures suggested in Willemsen (1975), we germinated seeds from maternal families from each population on damp filter paper in petri dishes with 1% plant preservative mixture. We placed the seeds in a germination chamber with a 24C day and a 18C night and a 14:10 light:dark cycle. After five to six days in the germination chamber, we transplanted the seedlings into one-inch pots that were filled with potting soil and were misted at regular intervals. The remaining seeds were monitored for another week and no further germination occurred after this point. After two weeks, we transplanted the seedlings to 3½ -inch pots with a 1/2 sand and 1/2 soil mixture. All plants were watered by automatically flooding the bench.
We randomly assigned the plants from each family to a treatment before transplanting. Individuals from each maternal family were assigned to two treatments (light stress and nutrient stress) and two controls (control 1 and control 2). Two weeks following the transplant, when the seedlings had approximately 4-8 true leaves, we harvested tissue from the first control (control 1). New leaves were collected, placed in a foil envelope, and instantly frozen in liquid nitrogen upon removal from the plant. All samples were collected in a random order. We then applied a light stress and a nutrient stress to the treatment plants. To simulate above ground neighbour effects, we constructed one shade box (1.5 m x 0.6 m x 1.3 m) with shade cloth and green filters to reduce the quantity and quality of light following Hodgins and Rieseberg (2011). To establish the nutrient stress, we flooded the trays with water that lacked the fertilizer, while all other trays received fertilizer. Treatments had a visible effect on the phenotype of the plants, particularly plant size. After two weeks of growth under stress conditions, we harvested three individuals per family in the two treatments and the remaining control (control 2) in the same manner as above.
We extracted RNA samples in a random order to avoid confounding batch effects with the experimental groups. We extracted total RNA using the TRIzol reagent (Invitrogen)/RNeasy (QIAGEN) approach as described in Lai et al. (2006). We used a Nanodrop to ensure that each sample had a concentration of >1.0ug/ul, a A260/A280>1.8, and a A260/A230>1.8.