The Molecular Mechanism Of Phase Change And Flowering In Precocious Trifoliate Orange (Poncirus Trifoliata) Via Suppression Subtractive Hybridization (SSH)

Citrus is one of the most economically important evergreen fruit crops all over the world. Flowering is an essential stage for fruit production, and thus it is important to understand the genetic mechanisms underlying the flowering event for genetic improvement. In 1976, a spontaneous mutant derived from Poncirus trifoliata (L.) Raf with short juvenile phase, namely, precocious trifoliate orange, was found in Yichang, Hubei province, China. Compared with 6 to 8 years of the wild-type trifoliate orange, almost all of the seedlings germinated from the mutant only have three years'juvenile period, and 20% seedlings even flowered in the year after germination. And the mutant seedlings can flower 2-3 times per year while the wild ones once per year. Because the mutant and wild-type had nearly the same morphology characteristics except flowering habit and no DNA polymorphism was detected between them, the mutant was speculated to be a direct variant of the wild-type, which is an ideal material for studying floral induction, inflorescence development, and flowering molecular mechanism. Suppression subtractive hybridization (SSH) and reverse Northern blotting were performed to decipher the early flowering development process of the mutant. The main results are as follows:1. In this study, differentially expressed genes between the mutant and wild type were identified by SSH and reverse Northern blotting. In juvenile-subtracted library, a total of 190 differentially expressed sequence tags (ESTs) related to flower development were identified. The temporal and spatial expression patterns of four SSH-enriched genes were studied in adult precocious trifoliate orange by real-time PCR, suggesting that these genes may play a critical role in the early flowering process of precocious trifoliate orange.2. In the adult-subtracted cDNA library,178 differentially expressed ESTs were obtained via the same methods. Among these ESTs, Y3E4, a sequence that shares high similarity with the Arabidopsis EARLY FLOWERING 5(ELF5) occurs frequently, Real-time PCR and in situ hybridization analysis proved that its expression pattern was closely correlated with floral induction, inflorescence development, and flowering. The full-length of Y3E4 was isolated by 5'and 3'rapid amplification of cDNA ends (RACE) and then transferred into Arabidopsis (ecotype Col-0) using the floral dip method. But the transgenic plants showed early flowering under long days, which is contrary to ELF5's characteristics in wild type Arabidopsis. This result demonstrates that ELF5 homologs might have evolved differentially between perennial woody and annual herbaceous plants.3. On the other hand, SSH was also performed to identify flowering-related genes between the mutant and the wild type in the juvenile phase. In the forward and reverse library,125 and 149 differentially expressed ESTs were identified respectively. The temporal and spatial expression patterns of selected ESTs were investigated. Of these ESTs, three genes (BARELY ANY MERITED, FLOWERING LOCUS T and TERMINAL FLOWER1) previously reported to be associated with or involved in developmental processes in other species were identified and further investigated by in situ hybridization and real-time PCR. The findings suggested that these genes may play important role during the early flowering process of precocious trifoliate orange.4. TERMINAL FLOWER 1 (TFL1)/CENTRORADIALIS (CEN)-like genes play important roles in determining plant architecture, mainly by controlling the timing of phase transition. In this study, we isolated and analyzed two TFL1/CEN-like genes from the early flowering mutant, PtTFL1 and PtTFL2. Real-time PCR determined that they were expressed in the vegetative tissues of both the adult and juvenile phases. In situ hybridization revealed that the two genes were also predominately expressed in the vegetative tissues. Constitutive over-expressing PtTFLl and PtTFL2 in Arabidopsis caused a late-flowering phenotype, indicating that TFL1/CEN gene family possesses conserved functions among various species.5. In Arabidopsis, MADS-box gene FLOWERING LOCUS C (FLC) is responsible for vernalization, which is a repressor of flowering genes. A MADS-box EST was isolated from an early flowering trifoliate orange mutant via SSH and the full length was obtained by RACE, which was designated as PtFLC. Phylogenetic analysis revealed that the MADS-box gene is more closely related to the homologs of the FLOWERING LOCUS C lineage than to any other MIKC-type MADS-box lineages known from Arabidopsis. Real-time PCR revealed that PtFLC was up-regulated in winter but down-regulated in spring and summer. Meanwhile, In situ hybridization showed that this gene was expressed in both the vegetative and reproductive meristems. In addition, our research also unraveled the involvement of alternative splicing in different developmental phases, and five alternatively spliced transcripts of the MADS-box gene were isolated. Expression analyses of these transcripts displayed that PtFLC1 and PtFLC2 were steadily detected in the juvenile period, whereas PtFLC4 and PtFLC5 were detectable only in the adult period and in various adult tissues. Compared with other spliced transcripts, the expression level of PtFLC3 fluctuated in juvenile tissue and could also be slightly detected in some adult tissues during a particular period. Based on these results, we presume that alternative splicing of PtFLC may adapt to the seasonal flower formation, cold-mediated dormancy breakage, and phase transition in precocious trifoliate orange.6. The total number of 31,468 unigenes from wild type and 31,468 unigenes from the mutant were isolated by massively parallel signature sequencing technology (MPSS). When the data from the two genotypes was combined, a total of 36,523 non-redundant unigenes were observed. Of these,6,859 were not observed in the wild type and 5,055 were not present in the mutant. The expression of 2,735 unigenes was significantly different at FDR<0.001 and│log2Ratio│≥1,1000 unigenes were up-regulated in the wild type as compared with the mutant. Meanwhile, the expressions of 1735 genes were decreased. In addition,30 flowering related miRNAs were isolated from the mutant by MPSS.