|Public on Jan 24, 2008
|Wild-type siblings in family segregating for T5-6b DpDf plants; Biological replicate 3
|Zea mays 11-day seedling aerial tissue; wild-type siblings in family segregating for T5-6b DpDf plants
|Genotype: Wild-type siblings (B73 background); Stage: 11-day seedling, all aerial tissue; Seed Source:49647-3self
|The T5-6b translocation, backcrossed into B73 maize genetic background for over 10 generations, was available from the University of Minnesota collection. The interchange T5-6b possesses a break at 5S.1 (ca. 300 cM on IBM2 2004 Neighbors genetic map) and between the middle and distal chromomere of the satellite of 6S (break occurs prior to 50 cM on IBM2 2004 Neighbors genetic map). Duplicate-Deficient (DpDf) heterozygous plants were identified among progeny derived from crossing a female B73/T5-6b translocation heterozygote by a male B73 plant. The chromosome constitution of DpDf heterozygous plants is normal for all chromosomes except 5 and 6. The DpDf plants contain one normal chromosome 6 and one 65 chromosome that is lacking the terminal chromomere of the chromosome 6 satellite and contains ~90% of the short arm of chromosome 5. Four biological replicates were grown using standard greenhouse conditions (1:1 mix of autoclaved field soil and MetroMix; 16 hours light and 8 hours dark; daytime temperature of 300C and night temperature of 220C) and sampled for gene expression on the 11th day after planting between 8:00 and 9:00 am. The plants were cut immediately above the highest brace root, thus all above-ground tissues and meristems were collected. For each biological replicate, sibling seeds produced by self pollination of a DpDf plant that segregate for wild-type and DpDf plants were planted individually and thirty plants were collected and genotyped using an SSR marker (bnlg161) that is tightly linked to the translocation breakpoint on chromosome 5. A pool of twelve wild-type plants and a separate pool of twelve DpDf plants were generated from each of the biological replicates. The sampled tissues were flash frozen in liquid nitrogen and stored at -80 0C prior to RNA isolation.
|For seedling RNA isolation, tissues from 12 seedlings/genotype/biological replicate were pooled and ground in liquid nitrogen. RNAs were extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Resuspended RNAs were purified further using the RNeasy system, according to the manufacturer’s instructions (QIAGEN, Valencia, CA).
|Eight ug of total RNA was labeled for each hybridization using the One-Cycle cDNA Synthesis Kit, according to the manufacturer’s instructions (Affymetrix, Santa Clara CA).
|Hybridization was performed according to the recommended Affymetrix protocols at the University of Minnesota Microarray facility.
|The Genechip 3000 scanner was used to scan each array
|No other relevant details
|MAS5.0 values are reported; the .cel file is also available
|Jan 23, 2008
|Last update date
|Jan 23, 2008
|Nathan M Springer
|University of Minnesota
|1445 Gortner Ave
|Profiling expression changes caused by a segmental aneuploid in maize