Adventitious roots (AR) develop from tissues other than the primary root, in a developmental process physiologically regulated by phytohormones. Adventitious roots provide structural support and contribute to water and nutrient absorption, and are critical for commercial vegetative propagation of several crops. Here we quantified the number of AR, root architectural traits and root biomass in cuttings from a pseudo-backcross population of Populus deltoides and Populus trichocarpa. Quantitative trait loci (QTL) mapping and whole-transcriptome analysis of individuals carrying alternative QTL alleles for AR number wereas used to identify putative regulatorsregions in the genome that regulateof AR development in the genome., and putative candidate genesregulators.
Overall design: Gene expression in three individuals carrying alleles that originated from the P. trichocarpa grandparent (UF352, UF498 and UF926, referred hereafter as the PtQTL genotype category) was contrasted with those with alleles from the P. deltoides grandparent (UF717, UF209 and UF912, or the PdQTL genotype category). 25 cuttings of each of the six selected genotypes (3 PtQTL and 3 PdQTL) were grown in hydroponic solution, and basal (1 cm) cutting sections from four biological replicates of each genotype were collected at each of five time points (0, 24, 48, 96 and 192 hours after cuttings were harvested). Total RNA was extracted from the bottom 1 cm stem section collected from each sample. The sample included xylem, phloem and bark. RNA was purified using RNeasy Mini Kit columns (Qiagen), and DNase treated with RNase-Free DNase set (Qiagen). RNA quality was evaluated in 1% w/v agarose gels. RNA was amplified and cRNA synthesized and labeled using Two Dyes Agilent Low Input Quick Amp Labeling Kit (Agilent). The microarray platform used consisted of single 60-mer probes designed for each of 43,803 predicted gene models from the sequenced genome of P. trichocarpa version 2. These probes were previously selected for being suitable for analysis of gene expression in this mapping population.
A total of 60 microarrays were used in the transcriptome analysis and each microarray was hybridized with 2 samples labeled with Cy3 or Cy5. Gene expression of each of six genotypes was analyzed in five time points (0, 24, 48, 96 and 192 hours), with four biological replicates per genotype and time point, following a “bird cage” design . The design was selected to favor contrasting gene expression of samples from different QTL categories (PtQTL and PdQTL) at each time point, as well as samples from the same genotype, collected from different time points. Median values of signal intensities were quantile normalized and log2 transformed. Normalized signals were analyzed in SAS 9.2 (SAS Institute Inc. 9.2® 2004, Cary, NC, USA) using a mixed-model ANOVA with genotype and genotype × time interactions as fixed effects, and microarray as random effect. Differences in expression between the group of genotypes from the PtQTL and PdQTL categories were estimated at each time point, and the significance was determined based on a false discovery rate (FDR) of 5%
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