lling requirements of earlier and later cultivars. A clus tering of these expression data is shown in Figure 2, with cultivars arranged according to their chilling requirements. In a previous work under our experi mental conditions, early cultivars Red Candem, Flor Red, May Glo, 86 6, Precocinho and Sunraycer required less than 412 chilling hours for dormancy release, intermediate cultivars Carolina and Crimson Baby needed 412 511 chilling hours, whereas Rose Diamond and Big Top showed requirements longer than 631 chilling hours. As expected in genes up regulated after dormancy Drug_discovery release, the overall gene expression was higher in early cultivars with low chilling requirements than in late cultivars with higher requirements.
Interestingly, the peach putative orthologs of Arabidopsis genes involved in pollen development programs were mostly grouped in two clusters, which argues for the existence of evolutionary conserved regulatory circuits orches trating the coordinated expression of these genes. Quantitative real time RT PCR confirm ation of microarray hybridization results allowed a more accurate determination of groups of similar expression. Eight genes from the cluster I of Figure 2 were analyzed by qRT PCR. All of them showed a common pattern, with higher and similar expression values in the cultivars Red Candem, 86 6 and Sunraycer, almost undetectable expression in Rose Diamond and Big Top, and intermediate values in the remaining five cultivars. On the other hand, ten genes analyzed from the cluster II showed a similar expression profile by qRT PCR, due to their higher transcriptional activity in Red Candem and Sunraycer.
The gene ppa011974m from cluster I and other five genes not included in clusters I and II in Figure 2 had a more gradual decline in expression from early to late culti vars, without drastic differences between cultivars with similar chilling requirements. We employed these qRT PCR data, based on their improved accuracy over microarray signals, to redefine two clus ters of coordinated expression in flower bud late genes, cluster A including IB153, PpB89, ppa020886m, ppa008548m, ppa018509m, ppa009789m , ppa021109m and ppa008777m, and cluster B containing ppa003797m, ppa006852m, ppa006506m, PpB71, ppa022178m, ppa019432m, ppa016810m , ppa011965m, PpB87 and ppa021373m.
The predominant expression in cultivars Red Candem, Sunraycer and to a lesser extent 86 6, indicates an earlier activation of genes involved in microsporogenesis and tapetum development in these cultivars. Flower bud late genes are transiently expressed in anthers The tissue specificity of genes belonging to clusters A and B was studied in the cultivar Big Top by qRT PCR. The transcript accumulation of these genes in vegetative buds was negligible when compared with their expres sion in flower buds, which precludes a general function of them in dormancy or growth resumption processes common to both vegetative and reproductive buds. Instead of that, flower bud lat