Zhang et al. BMC Genomics 2011, 12:63 http://www.biomedcentral.com/1471-2164/12/63 that the citrus genome contains orthologs to LFY. Accordingly, overexpressing the citrus LFY DNA sequence dramatically induced early flowering in trans- genic Arabidopsis [46]. Expression analysis of LFY by real-time RT-PCR showed that a transcript level of the gene was significantly up-regulated in the MT before the floral initiation had occurred (Figure 7). Therefore, we speculated that LFY is necessary to induce early flowering of precocious trifoliate orange. Citrus homo- logs for SOC1, FT, and AP1 were isolated previously and evaluated for their function in citrus or Arabidopsis [21,46,60,61]. They play important roles from the vege- tative phase to floral development in citrus. FT is a member of a small gene family in Arabidopsis that also contains TERMINAL FLOWER 1 (TFL1). Although TFL1 has been functionally characterized in citrus, we were not able to identify TFL1 sequences in this study. Our previous work also revealed that a large portion of the promotion of flowering by FT and TFL1 was achieved through down-regulation of TFL1 levels and up-regulation of FT levels in the MT [24]. One possible explanation for why these previously reported flowering- related genes were not discovered in our library is that their mRNA transcript levels were too low to be mea- sured by sequencing. In Arabidopsis, CO encodes a zinc finger protein that acts as a floral activator and mediates the photoperiod pathway, whereas FLC encodes a MADS box protein that acts as a floral repressor and mediates the autono- mous and vernalization pathways. CO and FLC regulate the expression of downstream genes, FT, SOC1, and LFY (Amasino 2010; Parcy 2005; Simpson and Dean 2002). We have identified seven unigenes showing sig- nificant (E-value lower than E-10) similarity to the Ara- bidopsis CO in two libraries. However, only a few of the CO-like genes were differentially expressed between the genotypes. Valverde et al. (2004) showed that the CO protein is ubiquitinated and then degraded by a protein complex called the proteasome, and that this process is regulated [62]. The autonomous and vernalization path- ways promote flowering by repressing FLC expression and many genes involved in the vernalization and auton- omous pathways control the epigenetic status of the FLC chromatin [14]. Although FLC homologous MADS box genes have been recently found from several eudicot lineages by phylogenetic analysis [63], FLC homologous genes have not been identified in any woody plants except citrus. Previous work in this mutant showed that specific splice variants of FLC were associated with tran- sition from juvenile to mature trees, and four alterna- tively spliced transcripts of FLC were isolated [22]. In this study, not only were alternatively spliced transcripts of putative FLC homolog found, but FRI homologs could also be found in the MT library and the WT Page 16 of 20 library. FLC was up-regulated in the MT library com- pared with the WT library, and the FRI expression pat- tern was contrary to that of FLC. Our previous work provided evidence that the expression profile of FLC was up-regulated during the winter, followed by a decrease in the spring and summer. This kind of cycling differs from the pattern observed in Arabidopsis [22]. A possible hypothesis suggests that due to the alternative splicing of PtFLC in citrus, which exerts its function in certain transcript form in the particular development stage, the total expression level of PtFLC was dispersed. Additionally, some sequences were found within the two libraries that would code for the other elements of the vernalization pathway: VRN1, VRN2, and VRN3 [64] or for the VIP3. VRN2 has a repressible role over the expression of FLC and codes for a protein with homol- ogy to PcG proteins [65]. These results indicate that the vernalization pathway may be present in citrus. Mean- while, these data also suggested that these genes may play a critical role in the early flowering process of pre- cocious trifoliate orange. Conclusions In this study, we used the MPSS method to monitor global transcriptional changes in the MT compared with the WT, and identified 2735 differentially expressed genes that were induced or repressed more than twofold in the MT at 0.05% significance level. MPSS data analy- sis uncovered a large number of genes that were not previously known to be involved in formation of the mutation trait. A number of new genes possibly related to flowering time were found in this study. In addition, we explored 110 putative components for the genetic flowering pathways in citrus by identifying homologs of Arabidopsis flowering-time genes. The expression of selected flowering-time genes corresponding to each flowering pathway were compared in the MT library and the WT library, most flowering-promoting genes had up-regulated expression in the MT library, while most repressors of flowering genes had down-regulated expression in the MT library. These data also indicate that all known genetic flowering pathways may be pre- sent in citrus. The function of these elements can now be tested in heterologous systems, such as Arabidopsis, via transgenic approaches. We believe our results will be a valuable source for future research on the control of flowering and of biennial fruit-bearing patterns in citrus. Methods Plant material and RNA preparation Wild-type and precocious trifoliate orange trifoliate orange samples were collected from the experimental fields of the National Citrus Breeding Center at Huaz- hong Agricultural University. The seeds of WT and