Zhang et al. BMC Genomics 2011, 12:63 http://www.biomedcentral.com/1471-2164/12/63 previously not known to be involved in the early flower- ing development process. Interpretation of the data soli- dified links between new information herein and our previous fragmentary knowledge, and provided new insight into the molecular processes regulating flowering time in citrus. Results Flowering characterization of WT and precocious trifoliate orange Self-pruning is a physiologic phenomenon in trifoliate orange in which shoots cease vegetative growth by auto- matically withering the shoot tip (0.5-1 cm, Figure 1b and 1e). Self-pruning is a necessary but insufficient con- dition for floral bud initiation. Until the late stage of self-pruning, the shoot apical meristem of juvenile tri- foliate orange is in an undetermined state and floral pri- mordia are not observed (October, Figure 1b). After self-pruning, the terminal bud and lateral buds of the juvenile trees enter dormancy until late February of the next year (Figure 1c). (In citrus, there are three flushes during the growing season. The spring flush is the most important one for growth and flower formation; these lateral buds develop into spring shoots in the next year.) A major characteristic of the MT is that its juvenile phase is shortened to 1 to 2 years, whereas the WT plant has a long juvenile period of 6 to 8 years. This “nonapparent” growth period is important for flower Page 3 of 20 induction and the transition from the vegetative to the flowering stage of precocious trifoliate orange. Cytologi- cal observation revealed that the floral buds in the pre- cocious mutant initiated differentiation immediately after self-pruning on spring shoots (Figure 1d-1f). Floral development hastened differentiation and produced the primordia of sepal, petal, stamen, and pistil sequentially. The whole flower bud integrates formation in one month, and then part of the flower bud population began to flower (Figure 1i-1m). However, most flower buds fell into dormancy until late February of the next year. For WT trifoliate orange, the spring shoots, which did not form floral buds, began to produce vegetative buds after self-pruning (Figure 1g and 1h). MPSS signature abundance and distribution MPSS libraries were constructed using RNA extracted from the terminal bud and the five following buds of spring shoots (after self-pruning) for the MT and the WT. A total of 16,564,500 and 16,235,952 successful reads were produced for MT and WT, respectively (Table 1). The sequence sets were filtered to remove low quality reads containing ambiguous nucleotides and adaptor sequences in both libraries, resulting in 16,067,565 (97.00%) clean reads for MT and 15,701,789 (96.71%) clean reads for WT (clean reads are termed as “signature” hereafter). The signature sets were filtered to remove any signatures that were Figure 1 Schematic diagram of developmental stages involved in the flowering of precocious trifoliate orange. a, Seedling of precocious trifoliate orange (1 month). b, The shoot apical meristem of seedling begins self-pruning (October). c, The terminal bud and lateral buds of the juvenile trees became dormant after self-pruning (mid-October). d, The lateral buds develop into a spring shoot in the next year (late February). e, The spring shoot begins self-pruning (early April). f, After self-pruning (mid-April). g, h, The lateral bud becomes a leaf bud (from late April to mid-May). i, Sepal primordia arise (late April). j, Petal primordia arise (late April). k, Stamen primordia arise (early May). i, Pistil primordia arise (mid-May). m, n, Full-developed floral bud (late May). o, p, The early flower morphology of precocious trifoliate orange.