Zhang et al. BMC Genomics 2011, 12:63 http://www.biomedcentral.com/1471-2164/12/63 7500 Real-Time System. Real-time PCR products were amplified with 1 μl of template of the RT reaction mix- ture, 10 μl of 2 × SYBR Green Master Mix, and 0.5 μl of forward and reverse primer (10 μmol/μl), with water to a final volume of 20 μl. The levels of gene expression were analyzed with ABI 7500 Sequence Detection Sys- tem Software (PE Applied Biosystems) and normalized with the results of b-actin. Real-time quantitative PCR was performed in four replicates for each sample, and data were indicated as means ± SD (n = 3). Additional material Additional file 1: Distribution of characteristic lengths of unigenes from the MT and the WT. Table 1: Length distribution of unigenes from the MT and the WT; Table 2: ORF length distribution of unigenes from the MT and the WT. Additional file 2: Expression of unigenes in the WT. The table lists sense and antisense expression genes of the WT. Additional file 3: Expression of unigenes in the MT. The table lists sense and antisense expression genes of MT. Additional file 4: Transcription factors found in the WT and the MT. Putative TF genes were identified by a BLAST search against rice, Arabidopsis, and Citrus sinensis TF genes downloaded from PlantTFDB (http://planttfdb.cbi.pku.edu.cn:9010/index.php). Additional file 5: The biological interpretation of the differential expression genes was further completed using KEGG pathway analysis. A total of 200 different metabolic pathways were found in this study, with some being consistent with biological processes already revealed by GO analyses. Additional file 6: Primers used for real-time quantitative RT-PCR for the verification of MPSS data. Optimal oligonucleotide sequences for real-time RT-PCR were predicted by primer express program to prevent faint PCR products as primer dimmer and false amplicon Additional file 7: The sequence information of citrus genes related to flowering development. Genes belonging to different flowering pathways are listed. Acknowledgements This research was supported financially by the National Natural Science Foundation of China (grant nos. 30971973, 30921002, 31071777), the Science Foundation of the Doctoral Discipline of the Chinese Ministry of Education (grant no. 20090146110009), the Fundamental Research Funds for the Central Universities (2010BQ044) and the 863 Project of China. Author details 1Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan 430070, PR China. 2National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, PR China. Authors’ contributions ZJZ, AXY, HCG, and SLM are responsible for generating the MPSS data and for interpretation of the results. ZJZ and ZDL carried out the RT-PCR experiments. ZJZ drafted the manuscript. HCG and ZJZ participated in research design and statistical analyses. GWW, DXX, and HCG proposed and supervised the research. All authors read and approved the final manuscript. Received: 11 August 2010 Accepted: 26 January 2011 Published: 26 January 2011 Page 18 of 20 References 1. Li C, Dubcovsky J: Wheat FT protein regulates VRN1 transcription through interactions with FDL2. Plant J 2008, 55(4):543-554. 2. Kikuchi R, Handa H: Photoperiodic control of flowering in barley. Breed Sci 2009, 59(5):546-552. 3. Jack T: Molecular and genetic mechanisms of floral control. Plant Cell 2004, 16(suppl_1):S1-S17. 4. Koornneef M, Alonso-Blanco C, Peeters AJM, Soppe W: Genetic control of flowering time in Arabidopsis. Annu Rev Plant Biol 1998, 49(1):345-370. 5. Putterill J, Laurie R, Macknight R: It’s time to flower: the genetic control of flowering time. Bioessays 2004, 26(4):363-373. 6. Boss PK, Bastow RM, Mylne JS, Dean C: Multiple pathways in the decision to flower: enabling, promoting, and resetting. Plant Cell Online 2004, 16(suppl_1):S18-S31. 7. Moon J, Lee H, Kim M, Lee I: Analysis of flowering pathway integrators in Arabidopsis. Plant Cell Physiol 2005, 46(2):292-299. 8. Kobayashi Y, Kaya H, Goto K, Iwabuchi M, Araki T: A pair of related genes with antagonistic roles in mediating flowering signals. Science 1999, 286(5446):1960-1962. 9. Kardailsky I, Shukla VK, Ahn JH, Dagenais N, Christensen SK, Nguyen JT, Chory J, Harrison MJ, Weigel D: Activation tagging of the floral inducer FT. Science 1999, 286(5446):1962-1965. 10. Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G: Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 2000, 288(5471):1613-1616. 11. Blázquez MA, Weigel D: Integration of floral inductive signals in Arabidopsis. Nature 2000, 404(6780):889-892. 12. Li D, Liu C, Shen L, Wu Y, Chen H, Robertson M, Helliwell CA, Ito T, Meyerowitz E, Yu H: A repressor complex governs the integration of flowering signals in Arabidopsis. Dev Cell 2008, 15(1):110-120. 13. Yoo SK, Chung KS, Kim J, Lee JH, Hong SM, Yoo SJ, Yoo SY, Lee JS, Ahn JH: Constans activates suppressor of overexpression of constans 1 through Flowering Locus T to promote flowering in Arabidopsis. Plant Physiol 2005, 139(2):770-778. 14. Amasino R: Seasonal and developmental timing of flowering. Plant J 2010, 61(6):1001-1013. 15. Helliwell CA, Wood CC, Robertson M, Peacock WJ, Dennis ES: The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. Plant J 2006, 46(2):183-192. 16. Blazquez MA, Green R, Nilsson O, Sussman MR, Weigel D: Gibberellins promote flowering of Arabidopsis by activating the LEAFY promoter. Plant Cell Online 1998, 10(5):791-800. 17. Brunner AM, Nilsson O: Revisiting tree maturation and floral initiation in the poplar functional genomics era. New Phytologist 2004, 164(1):43-51. 18. Talon M, Gmitter FG Jr: Citrus genomics. Int J Plant Genomics 2008, 2008:1-17. 19. Marques MC, Alonso-Cantabrana H, Forment J, Arribas R, Alamar S, Conejero V, Perez-Amador MA: A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus. BMC Genomics 2009, 10(1):428. 20. Pe aL, Martín-Trillo M, Juárez J, Pina JA, Navarro L, Martínez-Zapater JM: Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time. Nat Biotechnol 2001, 19(3):263-267. 21. Endo T, Shimada T, Fujii H, Kobayashi Y, Araki T, Omura M: Ectopic expression of an FT homolog from Citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Res 2005, 14(5):703-712. 22. Zhang JZ, Li ZM, Mei L, Yao JL, Hu CG: PtFLC homolog from trifoliate orange (Poncirus trifoliata) is regulated by alternative splicing and experiences seasonal fluctuation in expression level. Planta 2009, 229(4):847-859. 23. Zhang JZ, Li ZM, Liu L, Mei L, Yao JL, Hu CG: Identification of early-flower- related ESTs in an early-flowering mutant of trifoliate orange (Poncirus trifoliata) by suppression subtractive hybridization and macroarray analysis. Tree Physiol 2008, 28(10):1449-1457. 24. Zhang JZ, Li ZM, Yao JL, Hu CG: Identification of flowering-related genes between early flowering trifoliate orange mutant and wild-type trifoliate orange (Poncirus trifoliata L. Raf.) by suppression subtraction hybridization (SSH) and macroarray. Gene 2009, 430(1-2):95-104.