Zhang et al. BMC Genomics 2011, 12:63 http://www.biomedcentral.com/1471-2164/12/63 Page 12 of 20 Table 3 Citrus unigenes that share homology to flowering-time genes of other plants (Continued) U3656 U12668 U9906 U3220 U11760 U17573 U31888 U23754 U12575 U8652 U9909 U12495 U10522 2411 2514 4081 3148 1435 1158 713 732 1329 1325 2323 1047 1710 1156 1897 1684 854 168 162 50 15 71 1020 99 460 454 1400 1917 1828 790 143 161 41 11 81 1130 162 397 450 NM_180137 AJ011628.1 NM_101951 NM_180519 HM018601 XM_002517836 FJ502237 FJ502238 FJ502238 NM_180137 NM_105584 FJ373211 AB290727 534/715 (74%) 437/609 (71%) 1803/2671 (67%) 1221/1790 (68%) 335/469 (71%) 319/400 (79%) 595/639 (93%) 563/576 (97%) 1272/1326 (95%) 734/1115 (65%) 176/240 (73%) 428/435 (98%) 719/1066 (67%) 5E-130 0 0 2E-168 8E-62 5E-95 0 0 0 9E-86 1E-30 0 8E-88 SPL1 (Arabidopsis thaliana) SPL1(Arabidopsis thaliana) SPL14 (Arabidopsis thaliana) SPL7 (Arabidopsis thaliana) SPL10 (Vitis vinifera) SPL (Ricinus communis) SPL9 (Poncirus trifoliata) SPL13 (Poncirus trifoliata) SPL13 (Poncirus trifoliata) SPL1 (Arabidopsis thaliana) SPL6 (Arabidopsis thaliana) SVP (Poncirus trifoliata) TFL2 (Malus × domestica) several homolog genes belonging to the FRIGIDA (FRI) complex as well as other regulatory complexes (FRI- GIDA-ESSENTIAL1, SUPPRESSOR OF FRIGIDA4, and MINIDEPENDENS) involved in regulating the expres- sion of FLC. Also putative members of FLC repressing the PRC complex were present in the two libraries. These included putative VERNALIZATION INSENSI- TIVE 3 (VIN3), FERTILIZATION INDEPENDENT ENDOSPERM (FIE), EMBRYONIC FLOWER 1 (EMF1), VERNALIZATION 1 (VRN1), and LIKE HETEROCHRO- MATIN PROTEIN 1 (LHP1). In addition, a putative homolog for FLOWERING LOCUS D (FLD) sequence was identified in this study (Table 3); FLD plays a key role in regulating the reproductive competence of the shoot and results in different developmental phase tran- sitions in Arabidopsis. Lesions in FLD result in hypera- cetylation of histones in FLC chromatin, up-regulation of FLC expression, and extremely delayed flowering [45]. In addition to the photoperiod and the vernalization pathways, we searched candidate genes for autonomous and GA pathways. Several sequences corresponding to Arabidopsis genes from both pathways were identified, suggesting the presence of these pathways in citrus. Among these genes we found homologs for Arabidopsis FVE and SVP, which have been shown to control flow- ering in a specific thermosensory pathway. Moreover, some additional flowering-time regulators that have not been placed in any specific pathway were identified, such as the MADS transcription factor family genes, SQUAMOSA PROMOTER BINDING PROTEIN family genes, AGAMOUS family genes, and SEPALLATA family genes (Table 3). Differential expression of flowering genes between the MT and WT plants We compared the expression of selected flowering-time genes corresponding to each flowering pathway in the MT library and the WT library in order to explore the role of different pathways. Almost all flowering-related genes were differentially expressed between the genotypes; only a few of the genes were specifically expressed (Table 3). Of these, AP1 was not observed in the WT library, although a citrus AP1 had previously been identified [46]. The AP1 specifies flower meristem identity and is also required for normal development of sepals and petals. In the MT library, most flowering-promoting genes had slightly up- regulated expression compared with the WT library, such as FLD, PFT1, and SEP1. Floral integrator genes (FT, SOC1, and FLC) were clearly up-regulated in the MT library (Figure 6). On the other hand, most repressors of flowering genes were slightly down-regulated in the WT library compared with the MT library (Figure 6). Verification of the genes related to MT trait formation Transcriptional regulation revealed by MPSS data was confirmed in a biologically independent experiment using quantitative reverse transcription polymerase chain reaction (RT-PCR). A total of 30 genes were cho- sen to design gene-specific primers (Additional File 6); these genes included 26 significantly differentially expressed genes, two genes of no differential expression, and two genes encoding proteins previously reported to be associated with, or involved in, developmental pro- cesses in other species. The transcript abundance pat- terns of the MT and WT were compared with MPSS data. Results showed that for 28 of the 30 genes, real- time PCR revealed the same expression tendency as the MPSS data, despite some quantitative differences in expression level. Figure 7 showed differential expression levels for 28 genes (20 for induced, three for repressed, three for antisense, and two previously reported flower- ing-related genes) between MT and WT. For example, the floral integrator gene FT was up-regulated 1.7 times in MT compared with WT as analyzed by real-time PCR, consistent with MPSS data showing that the gene’s expression in the MT was threefold higher than in the