tissue: leaves phenotype: low photosynthetic rate genotype: F1 interspecific population (the female hybrid clone “YX01” (P. alba × P. glandulossa) and the male clone “LM50” (P. tomentosa))
Treatment protocol
Photosynthetic characteristics was collected from fully expanded leaves using the LI-6400 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA) at 9:00–11:30 on sunny days. Three functional leaves, the fourth to sixth counting from the individual stem head of all of the population were measured. According to the average of the data, fifty individuals which the value was in the peak were measured again to ensure the phenotype was stable. Among them leaves from thirty samples including fifteen individuals of high and fifteen individuals of low photosynthetic rate were selected to construct gene pool and immediately placed into liquid nitrogen in the field and stored at -80°C until used for isolating RNA.
Growth protocol
A total of 1200 progeny, which were randomly selected from the F1 interspecific population of 5000 pedigree, were used in this study. They established by controlled crossing between two elite poplar parents, the female hybrid clone “YX01” (P. alba × P. glandulossa) and the male clone “LM50” (P. tomentosa). The progeny was grown in Xiao Tangshan horticulture fields of Beijing Forestry University, Beijing, China (40°2′N, 115°50′E), using a randomized complete block design with three replications in 2008.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the Plant Qiagen RNAeasy kit (Qiagen China, Shanghai) according to the manufacturer’s instructions. Each replicate mixed RNA extractions of 5 individuals to equal quantities (2 mg) for later microarray analysis. All the RNA samples including every individual and the mixed integrities were examined by Agilent 2100 Bioanalyzer (Agilent Technologies, USA) according to the manufacturer’s instructions, and no evidence of degradation was noted. They subsequently were reverse transcribed into cDNA utilizing the SuperScript First-Strand Synthesis system and the supplied polythymine primers (Invitrogen).
Label
biotin
Label protocol
Total RNA were amplified, labeled and purified by using GeneChip 3’IVT Express Kit (Cat#901229, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instructions to obtain biotin labeled cRNA.
Hybridization protocol
Array hybridization and wash was performed using GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US)in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US)and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with default settings.
Description
Expression data from two years old leaves of F1 interspecific population Leaves from 5 low photosynthetic rate individuals.
Data processing
Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
