Zhang et al. BMC Genomics 2011, 12:63 http://www.biomedcentral.com/1471-2164/12/63 WT. Furthermore, the expression profile of six genes, including some citrus putative homologs for floral inte- grator or identity genes (SOC1 and AP1), and three other newly detected genes with significant transcrip- tional changes were analyzed at six stages during flower- ing development between the MT and WT (Figure 8). As expected, the expression of these genes was corre- lated with floral induction, inflorescence development, and flowering of precocious trifoliate orange. It is nota- ble that the expression levels of induced genes were up- regulated in the spring shoot apex samples of WT and MT genotypes before the floral initiation had occurred and repressed genes were down-regulated (Figure 8). Discussion We explored expression patterns at a specific stage of flowering development in an early flowering trifoliate orange mutant by MPSS. MPSS and ESTs can be used for quantitative measurements of gene expression when combined with genomic sequencing [28,29]. Both approaches describe similar patterns of transcript abun- dance, although there are some clear differences that are perhaps associated with the methods themselves. Com- pared with EST technologies, MPSS involves deep tran- script sampling and sequencing, usually 1-2 million transcripts per library, of a cDNA library on microbeads [29]. In principle, the MPSS data should provide a more thorough and quantitative representation of the absolute transcript population in terms of representation and relative abundance than EST data [28,30,47,48]. When compared with microarray technology that requires pre- vious knowledge of genes, the limitations to detecting unknown genes are not encountered in MPSS [48,49]. On the other hand, compared to microarrays, a high- throughput sequencing approach is technologically more complex, yet much simpler statistically and methodolo- gically [50]. The technology has been shown to provide comprehensive coverage and a sensitive measure of gene expression [51,52]. MPSS profiling has been used in stu- dies to address various biological questions, including a whole-genome transcript analysis in Arabidopsis [53] and in human [51], and transcript expression profiling of hybrid and inbred parents in oyster [54]. From the results of this study, it is apparent that MPSS analyses not only highlight some genes and biological processes already revealed by our previous macroarray data [23,24], but also reveal a large number of genes that are possibly involved in the formation of the early flowering trait. The data consistency from multiple approaches ensures that the MPSS data produced in this study are reliable. In addition, the approximately 3.6 × 108 base pairs of data produced here represent a substantial sequence resource and will contribute to genomic data available for citrus. Page 13 of 20 To understand the transcriptome profile and to isolate flowering-related genes during flowering development in citrus, genome-wide gene expression profiling was com- pared between WT and precocious trifoliate orange by using MPSS. The results revealed 2735 differentially expressed genes that were induced or repressed more than twofold in the MT at the 0.05% significance level. We identified a large number of newly discovered, intri- guing unigenes of transcription and post-transcription factors in these differentially expressed genes, indicating that these genes may be key regulators that control flowering development by activating or repressing numerous genes (Figure 7). Additionally, a number of putative homologs of genes for flowering time and floral organ identity were also found. To obtain additional insights into the functions of the differentially expressed genes, we examined the GO categorization for the most similar Arabidopsis homologs of each gene using func- tions within the TAIR website. Functional category ana- lyses revealed that a number of important pathways may work collaboratively in shaping the early flowering trait in the MT. Genes encoding proteins categorized as including binding (nucleotide binding, protein binding, and chromatin binding), biological regulation, and devel- opmental processes were enriched among those genes differentially expressed between the WT and the MT. We found a large proportion of “no homology” and similarity to unknown proteins sequences in the Gen- Bank database. The underlying genetics of flower induc- tion and floral organ formation may differ between woody perennials and herbaceous model species. Some of these genes may play important roles from the vege- tative phase to floral development in MT. In addition, a high proportion of antisense transcripts, i.e., 12,254 of 25,318 genes in MT and 11,892 of 22,510 genes in WT, were observed in our study. These results suggested that the early flowering trait could be regulated on transcrip- tional and posttranscriptional levels. In contrast, the MPSS data provided much more information for the regulation of these activities on the transcriptional level. A notable result is that a considerable percentage of the genes, 9% of the total annotated genes, were involved in the regulation of biological processes or transcription (Figure 5). The induction of regulatory genes of tran- scription correlates well with the increased overall tran- scription in the MT. The total number of transcribed genes in the MT was 31,468, more than in the WT (29,864), suggesting that newly initiated transcription occurs in the MT. Moreover, analyses of the genes with transcription changes greater than twofold showed that up-regulated genes constituted 63.4% of the total chan- ged genes. This suggests that these genes are related to uncharacterized mechanisms in the perception of signals and the initiation of flowering development.