Gene-specific forward and reverse primers, EjLFYfwd and EjLFYrev (Table 1), were designed based on the reported EjLFY-1 sequence (GenBank accession no. AB162033). Restriction enzyme sites were added additionally at the 5′-end and 3′-end of the primers for the subsequent vector construction. Flower bud and leaf of loquat cv. ‘Zaozhong No.6’ were used for the extraction of RNA and DNA, respectively. Full-length cDNA and DNA sequence of EjLFY-1 were obtained by PCR. PCR products were cloned into pGEM-T easy vector (Promega, Madison, WI, USA) and then sequenced.

To determine the involvement of EjLFY-1 in floral development of loquat, we detected its expression levels in different floral differentiation stages, which were generally grouped into four groups: vegetative buds, inflorescence buds, flower buds forming and blooming. Totally 12 samples (buds or flowers) were collected with 10-day intervals. 10 μg RNA from each sample was electrophoresed in 1.2% agarose gel and transferred to Hybond N⁺ nylon membranes (Roche, Swiss). A gene-specific fragment (498 bp) amplified by a pair of primers (DIGUP and DIGDW, Table 1) at the conserved region of its 3′ end was used as the probe for hybridization. The specific methods of probe labeling and hybridization are as described in the instruction offered by Roche DIG High Prime DNA labeling and Detection Starter Kit I (Roche, Swiss).

Full-length cDNA sequence of EjLFY-1 was subcloned into Xba I-Sac I sites of the binary vector pBI121 plasmid so that EjLFY-1 was under the control of CaMV 35S promoter (Figure 2). Recombinant plasmid pBI121-35S::EjLFY-1 was then introduced into the Agrobacterium tumefaciens strain EHA105.

Strawberry (Fragaria × ananassa) cv. Tudla was used for Agrotransfection. A. tumefaciens-mediated transformation was performed with the leaf-disk procedure (Nehra et al., 1990). Leaf segments were excised from in vitro plantlets grown on MS basal medium (Murashige and Skoog, 1962). The leaf segments were then cocultivated with A. tumefaciens harboring the plasmid pBI121-35S::EjLFY-1, followed by regeneration under the selection medium (MS+1.5 mg L^-1 TDZ+0.4 mg L^-1 IBA) containing kanamycin (30 mg L^-1). Regenerated buds were excised and transferred to 1/2MS medium containing kanamycin (50 mg L^-1) for rooting. Resistant plants from separate leaf explants were defined as independent transgenic events.

Genomic DNA were extracted from young leaves of kanamycin-resistant plants and the WT plants following a modified CTAB method (Ma et al., 2008), and then PCR analysis was carried out for detection of the insertion. Individual plants were tested for the presence of both EjLFY-1 and NPT II genes separately. To avoid the potential pollution caused by Agrobacterium may exist in the regenerated resistant plants, the existing of ChvA gene (Chromosomal virulence gene A) which specially belongs to Agrobacterium was also checked and excluded by PCR. Primers (AGRIF and AGRIR) and amplification methods are listed in Table 1.

For further verification of the integration of EjLFY-1, Southern blotting was performed using a randomly selected plantlet which had been confirmed by PCR. About 20 μg of genomic DNA was digested with EcoR I and then electrophoresed on a 0.8% (W/V) agarose gel. The digested DNA in the gel was then transferred to an Immobilon-Ny⁺ membrane (Millipore, USA). A fragment of NPT II gene obtained by PCR (primers: KANF and KANR, Table 1) was labeled as the probe using the DIG DNA Labeling and Detection Kit II (Roche, Diagnostics, USA). Hybridization was carried out according to the manufacturer’s instructions.

Non-transgenic strawberry (WT) was used as the control, except that the Agrobacterium strain containing recombined plasmid was used as a control for detection of the existence of ChvA gene.

Expression of EjLFY-1 in the first generation transgenic plantlet was detected by RT-PCR. Plantlets grown in greenhouse were used to extract the total RNA using an improved CTAB method as described by Chang et al. (2007). Before precipitation, the RNA was treated with RNase-free DNase I (Takara Biotechnology Co., Dalian, China) at 37°C for 4 h to eliminate the eventual genomic contamination. RNA integrity was assessed on a 1.0% agarose gel, and concentration was estimated using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The first-stand cDNA synthesis was performed with 1 μg total RNA and Oligo d(T)₁₈ primer using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the protocols provided by the manufacturers. Primers EjLFYfwd and EjLFYrev were used for the RT-PCR confirmation and 2 μl of the cDNA product was used as the template.

Quantitative real-time PCR (qRT-PCR) was performed to detect the expression of EjLFY-1 in the third-generation propagated clones originated from stolon buds of the transformants. Leaf was used as the material for the detection of EjLFY-1, shoot apex meristem was used to examine the expression level of FaAP1, a homolog of APETALA1 (AP1) in strawberry. Expression level of FaTFL1, homolog of the main floral repressor TERMINAL FLOWER 1 (TFL1) in strawberry, was also detected. Gene-specific primers used in qRT-PCR were also listed in Table 1. RNA extraction and cDNA synthesis were carried out following the method mentioned above. The qRT-PCR was performed using SYBR Green qPCR Kit (Takara Biotechnology Co., Dalian, China) following the manufacturer’s instructions. qPCR was repeated with three biological replicates, and each sample was assayed in triplicate by PCR. The 26S rRNA gene of strawberry was used as a internal control.

Independent transgenic lines and WT plants were rooted, transferred into pots and grown in a solar greenhouse in Shenyang Agricultural University (123°E, 41°N, Shenyang, Liaoning Province, China) at December of 2009. The agamic propagated plants from stolon buds of the transgenic plantlets were rooted and planted into new nutritional pots, and then separated from the donor plants.

At the end of each cropping seasons, old leaves and roots of the plantlets were partially removed and the plantlets were then transferred into a new nutritional pots separately.

The plantlets were successively flowering after growing in the greenhouse for 3–4 months and their horticultural traits were recorded individually. Flower timing of each sample was measured as the time period from transferring into pots to formation of the first flower. Plant height, flower numbers were also recorded.

The amino acid sequences were aligned and a phylogenetic tree was generated using the Clustal W method (Higgins et al., 1994). Boot strap values were derived from 1,000 replicate runs. The phylogenetic tree was constructed by MEGA 5.0 software (Tamura et al., 2011). Introns distribution diagram were drew with DNAMAN 6.0 software (Lynnon Biosoft, USA). The horticultural traits data were analyzed with EXCEL. Statistical analyses were performed using one-way ANOVA test, followed by Bonferroni’s test with SPSS software.

With a pair of gene-specific primers, a segment of 1,227 bp was isolated and sequenced. Sequence alignment showed that it is similar with the EjLFY-1 sequence which reported by Esumi et al. (2005). There are only three nucleotide acids and one amino acid diverse between the isolated sequence and the reported one, which may be used as molecular tool for cultivar differentiation. Alignment of the amino acids sequences encoded by EjLFY-1 and other LFY-like genes showed that they share the conserved domains with each other in the N-terminal and C-terminal, especially in the C-terminal conserve motif (Figure 3).

A phylogenetic tree was constructed by using the deduced protein of EjLFY-1 and the other reported LFY-like proteins with Neighbor-Joining Method (Supplementary Figure S1). EjLFY-1 falls in a small group constructed by other homologs of Rosaceae fruit trees, including the PpLFY of pear and the AFL1 and AFL2 of apple. These data are consistent with the traditional taxonomy, indicating their closely relationship during evolution process.

Full-length DNA sequence of EjLFY-1 was also obtained. Sequencing result showed that gEjLFY-1 (GenBank accession no. AY551183) was 2,602 bp in length, indicating the existing of intron(s). Alignment of gEjLFY-1 with its cDNA sequence showed the existing of two introns. The first intron is 464 bp and the second one is 911 bp in length. The amount of introns in gEjLFY-1 is also same as those reported LFY-like genes of other Rosaseae plants such as strawberry, dog rose and apple (Supplementary Figure S2).

Northern blotting data suggested that the transcription of EjLFY-1 initiated in the buds that still in morphologic vegetative stages (Figure 4, lanes 2–3), except in the earliest detected vegetative bud (Figure 4, lane 1). Transcription level of EjLFY-1 was increased gradually during the swelling period of the buds when the inflorescence meristems were initiated (Figure 4, lanes 4–6). Then the transcription level reached the topmost point when the inflorescence meristems started to generate flower meristems (Figure 4, lane 7). Once the flowers came into being, expression of EjLFY-1 decreased (Figure 4, lanes 8–9). No transcripts were detected in little flowers or blooming flowers (Figure 4, lanes 10–12).

An over-expression construct with EjLFY-1 CDS under the control of CaMV 35S promoter was introduced into strawberry cv. Tudla. Totally 59 resistant plantlets belonging to three independent lines (L1, L2, and L3) were got and rooted on MS medium containing kanamycin. The resistant plantlets and the WT ones were then transferred into pots and grown in greenhouse (Supplementary Figure S3).

To verify the genomic integration of the target gene, presence of EjLFY-1 and NPT II were confirmed by PCR separately. The expected specific fragments of both genes were detected in the randomly selected transformants (Figure 5A). While for the detection of ChvA gene, target fragment couldn’t be detected in any tested transformants (Figure 5A), indicating the detected fragments of EjLFY-1 or NPT II were caused by transformation event, excluded the possibility of Agrobacterium continuation.

L2-2, a random selected PCR-positive transgenic plantlet, was further tested by using Southern blotting detection for the integration of NPT II gene. Southern hybridization with the NPT II probe showed that two bands were visible in L2-2 while none was observed in the WT control (Figure 5B). This result provides a further evidence of the EjLFY-1 insertion in strawberry genotypes.

Expression level of EjLFY-1 in independent transgenic line was detected by RT-PCR (primers: EjLFYfwd and EjLFYrev, Table 1). Results showed that transcripts of EjLFY-1 could be detected in the tested transgenic lines (Figure 5C), but not in WT ones.

To evaluate the effect of over-expression of EjLFY-1 on the regulation of flowering process in strawberry, transgenic plantlets belonging to three independent lines (L1, L2, and L3) and the WT lines grown in small pots filled with nutrient soil were transferred into experimental greenhouse at December of 2009. It was found that plantlets belonging to L1 and L2 lines developed their inflorescences early and continuously at March of 2010, while the WT plants were still in vegetative growth at the meantime (Figures 6A–C). The early flowering habit of the transformants was very remarkable. Even at the April of 2010, when more than half plantlets of the L1 and L2 came into bloom, the WT ones didn’t develop inflorescences yet. Over-expression of EjLFY-1 promoted flowering process of the plants belongs to L1 and L2, their average flowering time were 23 and 41 days shorter than that of the WT, respectively (Figure 7). While for plants of line L3, the change of flowering formation period was negligible in compare with WT.

Other horticultural traits of the transformants were also tested at 2010. Over-expression of EjLFY-1 affected not only the flowering time of strawberry but also the flower quantities. Compared with WT plants, the transformants produced more flowers including the line L3 ones in which flowering time showed no significantly changes (Figure 7B). Even though, most flowers or fruits of the transformants showed no obviously changes compared with the WT ones, except that individual transgenic plantlets showing doubled sepals (Figure 6D).

Although over-expression of EjLFY-1 promoted the flowering process of the transgenic lines, it didn’t reduce the vegetative growth habit of those transformants at the first growing season. For example, three transgenic lines even showed a little higher average height phenotype than the WT ones at each investigated point except the beginning (Figure 7C).

Almost all the fruit species in commercial production must be propagated by asexual ways so as to keep their stable traits. Normally the nursery stock of strawberry is runner plant which comes from stolon bud of the donor plant.

Early-flowering habit was also maintained in the runner plants originated from L1 and L2. And with the continuous asexual propagation, the clones showed a more strong early-flowering phenotype: the third-generation runner clones came into flowering at a very young stage (Figure 8A). And unlike the almost unchanged vegetative growth habits of the transformants in the first cropping season, runner clones showed an obviously reduction in vegetative growth. For example, plant showed a dwarf phenotype and their leaf were also extremely smaller than those of the WT ones (Figure 9).

Flowering of the runner clones were not only early but also continuously. Some runner plantlets of the transformants even could develop into flower in the hot summer, when the WT ones were still in their vegetative growth phase and only produced stolons (Figure 8C). In addition, variation of flower organs was also found in individual plantlets of the progeny, such as the multiplicated sepals (Figure 8B) in one runner plantlet originated from L1.

To investigate whether the effect of ectopic expression of the transgene could be maintained in their asexual propagule, expression of EjLFY-1 was also detected in the runner plants originated from the transformants.

qRT-PCR detection showed that expression of EjLFY-1 also existed in the third-generation runner plants originated from the transformants, although the expression levels varies (Figure 10). As the downstream target gene of LFY homologs, expression of FaAP1, the AP1 homolog in strawberry, was also detected. Results showed that although the EjLFY-1 expression were higher in all the transgenic runner plants than that of the WT ones (in which were zero), expression of FaAP1 in those plants differed: its expression in OXRP-1 (Over-expression runner plant 1) was higher than not only the WT ones but also the OXRP-2 and OXRP-3 (Figure 10). The varied expression levels were also detected in FaTFL1, the main repressor in flowering process (Figure 10). Generally, the FaTFL1 expression in WT was lower than those in all the three tested transgenic runner plants.