Zhang et al. BMC Genomics 2011, 12:63 http://www.biomedcentral.com/1471-2164/12/63 promotion pathways are integrated through the tran- scriptional regulation of two “flowering-time” genes, FLOWERING LOCUS T (FT) [8-10] and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and two floral meristem identity genes, LEAFY (LFY) and APETALA1 (AP1) [10-13]. Among the integrators, FT and SOC1 have a more direct function in determining flowering time. They share the common upstream regu- lators CONSTANS (CO), a key component in the long day pathway, and FLOWERING LOCUS C (FLC), a flow- ering repressor integrating autonomous and vernaliza- tion pathways [10]. FLC suppresses flowering, at least in part, by repressing the expression of the floral activators SOC1 and FT, and it has been shown to bind directly to these activators [14,15]. LFY is a key player in the speci- fication of floral meristem identity [16], and it dramati- cally increased the number of coflorescences in a lfy mutant due to the activity of AP1 [7]. Thus, crosstalk between pathways might explain how the multiple sig- nals affecting flowering are coordinated, and differences in how pathways are integrated might underlie the diversity of plant flowering [3,6,17]. Regulation of flowering in woody perennials shows remarkable differences with respect to herbaceous spe- cies. Annual plants complete their life cycle in one year and initiate flowering only once, whereas most other fruit crops have a long juvenile period, during which no reproductive development occurs. After this period, however, flowering happens repeatedly. How perennial woody plants undergo a long silent juvenile stage and then repeated vegetative growth and flower- ing has not been extensively studied at the level of transcription [17]. Citrus is one of the most widespread fruit crops in the world, with great economic and health value [18]. It is among the most difficult plants to improve through tra- ditional breeding approaches due to undesirable repro- ductive traits and characteristics [19]. These include degrees of sexual sterility and incompatibility, nucellar embryony, extended juvenility, and large plant size. To accelerate flowering time, constitutive expression of LFY or AP1 derived from Arabidopsis was used to dramati- cally reduce generation time in citranges [20]. In a simi- lar approach, but with a citrus gene, it was shown that transgenic poncirus carrying the CiFT (an FT homolog) also exhibited early flowering, although this phenotype was accompanied by several pleiotropic effects [21]. In addition, the long juvenile phase (6 to 8 years) of most citrus genotypes impedes the rapid evaluation of trans- genic trees modified to affect adult traits such as fruit quality (e.g., rind and flesh color, flavors, maturity dates, seediness, abscission, peelability, rag content, acid and sugar levels) and other traits associated with productive mature trees. Thus, understanding the molecular Page 2 of 20 regulation of the flowering process is crucial for control- ling fruit production in citrus. In 1976, precocious trifoliate orange with a short juve- nile phase, a spontaneous mutant (MT) derived from Poncirus trifoliata (L.) Raf, was found in Yichang, Hubei province, China. In contrast to the 6- to 8-year juvenile period of the wild-type (WT) trifoliate orange, almost all of the seedlings that germinated from the MT had only a 3-year juvenile period; 20% of the seedlings even flow- ered in the year after germination. The MT seedlings can flower two to three times per year, while the WT plant flowers only once per year. The MT and the WT had nearly the same morphological characteristics aside from flowering habit, and no DNA polymorphisms were detected between them. Consequently, the MT was speculated to be a direct variant of the WT, which was ideal for studying floral induction, inflorescence devel- opment, and the flowering molecular mechanism. Therefore, a transcriptional study including a cDNA macroarray in combination with suppression subtraction hybridization (SSH) was used to investigate gene expres- sion changes in the MT, and a total of 368 differentially expressed genes were detected [22-25]. Interestingly, most of the 368 genes showed differential expression in the year after self-pruning, indicating that this period is a critical stage for the transcriptional regulation of the MT trait formation. Taken together, our previous research using SSH technology has provided important clues for understanding the formation of the mutation trait in precocious trifoliate orange; however, the tran- scriptional information from SSH, especially for genes expressed at low levels, is rather limited. Further ana- lyses of gene expression during the early flowering pro- cess is needed. Massively parallel signature sequencing technology (MPSS), like expressed sequence tags (ESTs) and serial analysis of gene expression (SAGE), is a sequence-based method that can be used for transcriptional profiling to measure gene expression. However, MPSS provides more thorough qualitative and quantitative descriptions of gene expression due to its tremendous depth [26-30]. This depth enables identification of a nearly complete inventory of transcripts in a given sample [31]. To date, the MPSS method has provided a rapid way to identify and profile differentially expressed genes in a variety of plants, mutants, and tissues, and at different stages of development [28,30-32]. In this study, a genome-wide gene expression study was carried out between WT and precocious trifoliate orange by using MPSS, and a total of 36,523 genes were analyzed. Of these genes, 2735 showed a twofold or greater expression difference between the MT and WT. The results demonstrated that some genes may be newly transcribed in the MT. Our results also identified a large number of genes